System and method for identifying blood vessels during fluorescence imaging

Abstract

Blood vessels in the tissue are continuously identified by fluorescence imaging.SOLUTION: A computer-implemented method for identifying a blood vessel in a tissue of a subject includes generating and / or receiving a fluorescence image of the tissue in which a fluorescence signal oscillates in a predetermined pattern, and analyzing the fluorescence image and the associated oscillation signal to identify the blood vessel in the tissue.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to systems and methods for continuously identifying blood vessels in tissue by fluorescence imaging. In particular, the present disclosure relates to continuously measuring and assessing hemodynamics in tissue during medical procedures using fluorescence imaging, where the administration of a fluorescent agent is controlled and automated, thereby identifying, mapping, and visualizing blood vessels in the tissue. [Background technology]

[0002] background Tissue dissection is a large part of surgical procedures today, often occupying up to 50% of operative time. Great attention is paid to separating blood vessels and other central structures from the surrounding connective tissue before the actual surgical procedure is performed. This is the case in surgical procedures such as bowel resection and anastomosis. Despite this, however, unintentional rupture of blood vessels frequently occurs, significantly prolonging operative time and resulting in excessive bleeding and other complications.

[0003] The challenge is that, to the naked eye, blood vessels and central structures are not easily distinguishable from the surrounding connective tissue. And the problem is exacerbated with the increasing prevalence of obesity, because obese patients have a high amount of connective tissue, primarily composed of fat. Therefore, surgeons need tools that can easily identify and map blood vessels in connective tissue. This allows for safer and faster tissue dissection, potentially substantially reducing surgical time. Shorter surgical times are beneficial to the patient (who is exposed to less surgical stress) and the hospital as a whole, because it allows for more surgical procedures in the same time period.

[0004] Fluorescence imaging is one such tool in which injection of a fluorescent imaging agent (also known as a fluorescent contrast agent, also known as indocyanine green (ICG)) is provided to visualize blood flow and perfusion in tissues and anatomical structures. One recent example is the Leica Microsystems GLOW800 augmented reality (AR) system, which provides simultaneous white light and real-time fluorescent blood flow views, so that brain anatomy can be observed in natural color and augmented by real-time blood flow. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention One limitation of known fluorescence imaging procedures is that, for example, typical doses of ICG used today require at least 20–30 minutes of washout before a new measurement can be taken; that is, each dose of ICG can be considered a “single image exposure” because the bolus flows through the artery and provides a snapshot of the perfusion situation. The problem is that during intestinal surgery, the intestine is prone to peristalsis, and during tissue dissection, the surgeon works slowly through the connective tissue; therefore, a single snapshot every 30 minutes is hardly usable. To overcome this limitation, the inventors of pending PCT application PCT / EP2019 / 065648, entitled “System and method for automatic perfusion measurement,” present a novel administration regimen for fluorescence imaging that enables “continuous” perfusion monitoring based on the automated administration of time-separated microboluses of at least one fluorescent agent. This approach produces a fluorescent signal from the perfused tissue that fluctuates over time in a predetermined pattern. PCT / EP2019 / 065648 is incorporated herein by reference in its entirety. It is used as a reference.

[0006] The present inventors have now realized that the novel administration regimen disclosed in PCT / EP2019 / 065648 can be applied to identify blood vessels (e.g., blood vessels hidden in connective tissue). Accordingly, one aspect of the present disclosure relates to a (computer-implemented) method for identifying blood vessels in tissue of a subject, for example, during a medical procedure. The method comprises continuously generating and / or receiving fluorescent signals from tissue (from blood vessels in the tissue) in which the intensity and / or wavelength of the fluorescent signal fluctuates and / or oscillates in a predetermined manner (e.g., oscillates in a predetermined pattern). That is, the oscillating pattern is preferably externally imposed in a predetermined and controllable manner. The oscillating pattern is preferably generated from a series of small boluses of at least one fluorescent imaging agent. That is, the oscillating pattern results from repeated bolus injections. The oscillating pattern can be understood to have a constant frequency (also known as a period). As a result, a series of boluses administered with a defined and / or controlled duration between subsequent boluses determines the frequency of the pattern.

[0007] The unique time difference in each of the fluorescent signals from each small bolus due to subject-specific hemodynamics can be used to identify blood vessels. That is, the disclosed approach analyzes at least a portion of the fluorescent image and identifies the following: the time lag between the bolus injection and the arterial or venous fluorescent signal; the time difference between the arterial and venous fluorescent signals; the time difference between the arterial or venous fluorescence signal and the tissue fluorescence signal; determining at least one time difference selected from the group consisting of: may include:

[0008] Alternatively, the approach can be formulated as continuously generating and / or receiving fluorescent signals from tissue (from blood vessels in the tissue) where a phase difference in the fluorescent signals from blood vessels and surrounding tissue is repeatedly and / or sequentially imposed.

[0009] As disclosed in PCT / EP2019 / 065648, one way to provide an oscillating fluorescent signal is to inject microboluses of ICG at regular intervals over an extended period of time, such that the intensity of the fluorescent signal oscillates at a predetermined frequency, i.e., each time a bolus is injected, the fluorescent signal subsequently appears, the frequency of oscillation being determined by the time of bolus injection. As exemplified herein, microboluses allow for continuous injection of ICG at controllable, predetermined and / or regular intervals over a period of one hour or even several hours, e.g., at regular intervals of 1-10 minutes, e.g., 1-2 minutes, 2-3 minutes, 3-4 minutes, 4-5 minutes, 5-6 minutes, 6-8 minutes, or 8-10 minutes.

[0010] Since the fluorescent signal from the small bolus continues to come again and again, the blood vessel can thereby be identified again and again in the fluorescent image of the tissue, ie, based on the time difference and the oscillating fluorescent signal.

[0011] That is, the disclosed method may consequently further comprise the step of continuously acquiring fluorescence images of the tissue, preferably similarly analyzing said fluorescence images and their associated fluctuating and / or oscillating intensity, wavelength and / or phase, thereby continuously identifying blood vessels in the tissue.

[0012] There are a variety of known methods for analyzing fluorescent signals and thereby visualizing, for example, blood vessels. An important aspect of the previously disclosed approaches is the ability to visualize when exploring arteries, veins, tissue, etc. Repeated injections of small boluses ensure that fluorescent signals are repeatedly present for analysis, and determination and / or recognition of subject-specific hemodynamic time constants ensures knowing when to look for or when to anticipate signals originating from arteries, veins, tissues, and / or other sources.

[0013] One or more subject-specific hemodynamic parameters may be known in advance (e.g., one or more hemodynamic time constants), e.g., from a previous procedure. Thus, the present disclosure also relates to a method for identifying blood vessels in a tissue of a subject, e.g., during a medical procedure, the method comprising: - successively acquiring fluorescent images of tissue in which the fluorescent signal oscillates in a predetermined pattern having a constant frequency, said pattern preferably being generated from a series of boluses of at least one fluorescent imaging agent, said series of boluses being administered with defined and / or controlled durations, with successive boluses determining the frequency of said pattern; - analyzing at least some or all of the fluorescent images; and - identifying blood vessels in the fluorescent image of said tissue, preferably continuously and / or repeatedly, based on said subject-specific hemodynamic parameters and on the fluorescent signal oscillating in a predetermined pattern; Includes.

[0014] A further aspect of the present disclosure relates to a system for identifying blood vessels in tissue during a medical procedure on a subject, the system being configured to continuously generate fluorescent signals from blood vessels in the tissue, the fluorescent signals oscillating in intensity in a predetermined pattern, continuously receive fluorescent images of the tissue, and analyze the fluorescent images and their associated oscillation intensities, thereby continuously identifying blood vessels in the tissue. The system may include a controllable injection pump for holding at least one first fluorescent imaging agent, the injection pump configured to inject a series of defined boluses of the first fluorescent imaging agent into a vein of the subject, thereby generating the fluorescent signal oscillating in the predetermined pattern. The system may accordingly be configured to perform the methods disclosed herein.

[0015] The identified vessels can then be visualized and mapped to medical personnel, for example, before and during tissue dissection. A key advantage is that the disclosed approach, i.e., automated and continuous vessel identification and detection, can be performed in the background while the surgeon works while viewing the normal white-light camera image. For example, a microbolus procedure can be initiated at the beginning of surgery and run in the background, monitored by a computer system that measures and receives fluorescent signals and controls the interval and administration of the microbolus regimen. The surgeon can then shift to a computer-generated "vascular view" showing the vessels in the region of interest at any time during surgery. Advantageously, the continuously identified vessels can be superimposed onto the white-light image in real time, so that otherwise hidden vessels appear in the white-light image as augmented reality objects in real time.

[0016] The approach disclosed herein can be of great use to surgeons during surgery, in any organ, and for any indication, especially when vascular structure and anatomy may be important. It can also be used in plastic surgery, for example, to evaluate the vascular structure and anatomy of a skin flap to be transplanted. In that case, it can provide information about where to excise the flap. Similarly, it can provide important information when the flap is transplanted into a recipient, in which case the approach can provide information about the vascular structure and the success of the vascular anastomosis.

[0017] The disclosed approach used within a surgical procedure can be seen as a surgical decision support. However, the use of the disclosed approach is not limited to use during a surgical procedure. The disclosed approach can also be advantageously applied to provide information about vascular structure and anatomy before or after surgery, or even for monitoring wound healing and vascular anatomy in patients who have not undergone surgery at all. In such cases, the disclosed approach can be viewed as a physical examination tool, similar to a CT scan.

[0018] The present disclosure further relates to a computer program (product) having instructions that, when executed by a computing device or computing system, cause the computing device or computing system to perform a method for identifying blood vessels in tissue of a subject as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1A shows an example of an intensity curve after a bolus of ICG was provided to a subject, and FIG. 1B shows the corresponding intensity curve in which the hemodynamic parameters perfusion slope, slope start, slope end, maximum intensity, washout slope, washout start, and washout slope end were calculated and plotted.

[0020] [Figure 2] FIG. 2A shows an oscillatory time intensity fluorescence curve in which oscillations are disrupted due to the onset of ischemia in a human subject.

[0021] FIG. 2B shows an expansion of the time interval around t=3800 seconds of the graph before the onset of ischemia occurs.

[0022] FIG. 2C shows idealized data with and without ischemia.

[0023] FIG. 2D shows idealized data in which only a portion of the oscillating time-intensity fluorescence curve can be detected.

[0024] [Figure 3] FIG. 3A shows serial measurements of a human subject injected with a microbolus.

[0025] FIG. 3B shows an expanded view of the interval shown in FIG. 3A.

[0026] [Figure 4]FIG. 4 shows measurements in a human subject to venous occlusion, where blood flow is only partially restricted.

[0027] [Figure 5] Figure 5A shows a snapshot of an ICG analysis running on a humanoid subject (right forearm), taken at a very early stage when a microbolus of ICG has just been administered and is beginning to enter the arteries.

[0028] FIG. 5B shows a snapshot taken a few seconds later than FIG. 5A, in which multiple arteries can be identified.

[0029] [Figure 6] FIG. 6A shows a snapshot taken a few seconds later than FIG. 5B, when the fluorescence intensity from a particular microbolus of ICG is reaching its peak value.

[0030] FIG. 6B shows a snapshot approximately one minute later than FIG. 6A, in which veins can be identified. The washout phase is illustrated.

[0031] [Figure 7] FIG. 7 shows an edge-filtered version of FIG. 6B.

[0032] [Figure 8] FIG. 8 shows an image in which an artery is identified in the sequence of images shown in FIGS. 5A-6B.

[0033] [Figure 9] FIG. 9 shows images in which veins have been identified in the sequence of images shown in FIGS. 5A-6B.

[0034] [Figure 10] Figure 10 shows an image in which the arteries and veins shown in Figures 8-9 have been visually enhanced in red (arteries) and blue (veins), and the images have been superimposed so that they are easily distinguishable. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description One embodiment of the present disclosure relates to a computer-implemented method for identifying blood vessels in a tissue of a subject, for example, during a medical procedure, the method comprising the steps of: continuously receiving fluorescent signals from blood vessels in the tissue, the fluorescent signals oscillating in intensity in a predetermined pattern; continuously acquiring fluorescent images of the tissue; and analyzing the fluorescent images and their associated oscillation intensities, thereby continuously identifying blood vessels in the tissue; Includes.

[0036] The predetermined pattern can be characterized by a defined frequency with a period of 30 to 15 seconds, e.g., between 1 and 10 minutes, or between 1 and 2 minutes, 2 and 3 minutes, 3 and 4 minutes, 4 and 5 minutes, 5 and 6 minutes, 6 and 8 minutes, or 8 and 10 minutes, over a period of at least 10 minutes, or at least 15 minutes, or at least 30 minutes, or at least 1 hour, or at least 2 hours, where the fluorescent signal oscillates in intensity according to the predetermined pattern. As also described herein, the predetermined pattern can be derived from the controlled injection of a series of small boluses of at least one fluorescent agent (e.g., ICG).

[0037] In a preferred embodiment, the identified vessels are continuously displayed and visualized, for example, on a screen. Additionally, the identified vessels may be combined to identify one or more networks of interconnected vessels.

[0038] White light images of the tissue can also be continuously received and acquired, so that at least one white light image of the tissue can be generated (in which the identified blood vessels are visually enhanced, for example, by superimposing the identified blood vessels onto the white light image and preferably also visually enhancing the blood vessels, for example, with high contrast colors) and displayed on a screen so that the identified blood vessels appear as augmented reality objects.

[0039] A preferred embodiment of the disclosed approach includes identifying arteries and veins in the identified vessels, and this identification between arteries and veins is advantageously based on a predetermined pattern of oscillating fluorescent signals. Bolus hemodynamics differs in arteries and veins, for example, when a bolus of ICG spreads through a patient, and the fluorescent signal appears first in the arteries, then in the microcirculation in the surrounding tissue, and some time later in the veins. Thus, the time difference between the bolus injection and the arterial, tissue, and venous signals can be determined from the first or first few boluses, for example, by analyzing a series of corresponding images, looking for signals in that order. The novel microbolus dose regimen employed superimposes numerous time and / or wavelength dynamics that can be exploited. Because only one or a few microboluses of contrast agent are required to determine patient-specific / situation-specific time differences between the bolus injection and the arterial signal, and between the arterial and venous signals, these time differences can be exploited in subsequent controlled microbolus administrations to sequentially differentiate arteries and veins. The time differences between the arterial and tissue signals, and between the tissue and venous signals, can also be exploited. The washout period of each microbolus also involves different hemodynamics of arteries, veins, and surrounding tissues, which can be exploited to identify vessels and differentiate between arteries and veins, even during the washout period between bolus injections.

[0040] One example of identifying blood vessels in a fluorescence image is by image filtering, preferably edge filtering, of the acquired fluorescence image. Due to the oscillating fluorescence signal and hemodynamics in the tissue, there are almost always regions in the acquired image where the image brightness changes sharply, i.e., has discontinuities. These regions are most often either arteries or veins; therefore, by consistently applying appropriate image filtering, blood vessels appear substantially constant during successive bolus administrations.

[0041] Another, more accurate approach is to identify blood vessels based on the inherent phase difference between the fluorescent signals associated with each blood vessel and surrounding tissue. This is due to hemodynamics in the subject / patient's body. As disclosed herein, the global oscillating fluorescent signal is imposed by controlled and repeated injections of small boluses of a fluorescent agent, such as ICG. Each bolus of fluorescent agent also produces a fluctuating fluorescent signal at a more local level due to hemodynamics in the subject. Each small bolus of fluorescent agent reaches arteries, tissues, and veins at different times, and the disclosed approach utilizes these time differences to identify blood vessels, distinguish between blood vessels and tissues, and distinguish between arteries, veins, and tissues. The time difference between the fluorescent signals originating from arteries, tissues, and veins can be seen as a phase difference in the sequential evolution of the fluorescent signals. That is, at any time during the oscillating fluorescent signal, there is a phase difference at different locations in the corresponding acquired fluorescent image due to the inherent time difference resulting from the fluctuating hemodynamics in the arteries, veins, and surrounding tissues, i.e., the subject's hemodynamics. The subject-specific time difference may be known or the first of several boluses may be used to determine the subject-specific time difference, i.e., to determine at least one time difference selected from the group consisting of:

[0042] the time difference between the bolus injection and the arterial or venous fluorescence signal, the time difference between the arterial and venous fluorescence signals, and the time difference between the arterial or venous fluorescence signal and the tissue fluorescence signal. One or more of these time differences can be "translated" into a corresponding phase / phase difference or one or more of blood vessels, arteries, veins, surrounding tissue, and / or something else. Thus, knowing the expected phases of the arteries, veins, and surrounding tissue, it is possible to associate each pixel, or ROI, in the fluorescence image with a classification of either artery, vein, surrounding tissue, or something else.

[0043] The predicted phase of the arteries, veins, and surrounding tissues at a given time point can be directly related to a predetermined oscillation pattern that determines the period and oscillation frequency of the oscillating fluorescent signal. The predicted phase can either be estimated, calculated, and / or determined during a medical procedure, for example, as an initial learning phase in which the oscillating fluorescent signal is observed for one or several microboluses to measure its associated hemodynamics in a specific situation. The predicted phase of the arteries, veins, and surrounding tissues can thereby be determined based on the period and oscillation frequency of the oscillating fluorescent signal. Thus, in a subsequent medical procedure, the time of each bolus injection provides information about the expected phase of the arteries, veins, and surrounding tissues.

[0044] Thus, a preferred embodiment of the disclosed approach involves determining the phase difference between a fluorescent signal originating from an artery in the tissue, a fluorescent signal originating from a vein in the tissue, and optionally a fluorescent signal originating from tissue surrounding the blood vessel, and relating this phase difference to a predetermined vibration pattern.

[0045] Thus, analyzing the phase and / or phase difference in a sequence of fluorescence images is one way to identify blood vessels in a fluorescence image. That is, blood vessels can be identified / detected in a fluorescence image sequence at a pixel-by-pixel level by knowing the phase of the fluorescence signal—and this can be detected at any time during a microbolus dose regimen. This phase information, combined with knowledge of the given bolus dose (i.e., that its fluorescence signal oscillates in a predetermined pattern), provides the necessary information as to whether a pixel in an image, or a region of interest, is a blood vessel, and further whether a pixel in an image, or a region of interest, is an artery, a vein, tissue, or something else. This allows for the actual mapping of blood vessels (including arteries and veins) to be provided, for example, pixel-by-pixel, or group of pixels, or ROI-by-ROI, with each pixel—or ROI—essentially classified as a blood vessel (preferably including an artery or a vein), surrounding tissue, or something else, as needed, in each fluorescence image.

[0046] Taken all together, the disclosed approach can provide medical personnel with a constantly updated map of blood vessels (including arteries and veins) in the surgeon's field of view / anatomical region of interest. In particular, the identified arteries and veins can be superimposed onto a white light image, visually enhanced so that the arteries and veins are visually distinguishable (e.g., each has a distinct high-contrast color), and displayed on a screen.

[0047] Once identified and possibly mapped in a fluorescence image, or sequence thereof, the vessels (preferably including the identified arteries and veins) can be tracked in subsequent fluorescence images, even if motion (e.g., peristalsis) is involved. Tracking of the identified vessels can be provided, for example, by using tracking methods available in the prior art and known to those skilled in the art. An example of tracking in fluorescence images is disclosed in WO 2018 / 104552.

[0048] In a further embodiment, the method of the present disclosure further comprises administering a series of boluses of at least one fluorescent imaging agent into the subject (intravenously), thereby generating a predetermined pattern of oscillating fluorescent intensity and / or wavelength, the series of boluses preferably being administered with a defined duration between successive boluses.

[0049] The preferred fluorescent imaging agent used is ICG, with each bolus of ICG preferably corresponding to less than 0.01 mg ICG / kg body weight of the subject, more preferably less than 0.005 mg ICG / kg body weight, even more preferably less than 0.004 mg ICG / kg body weight, even more preferably less than 0.003 mg ICG / kg body weight, even more preferably less than 0.002 mg ICG / kg body weight, and most preferably less than 0.001 mg ICG / kg body weight. The series of boluses may be injected automatically by a controllable, e.g., computer-controlled, injection pump.

[0050] The imaged tissue may be part of an internal organ of the subject. The imaged tissue may also be part of an anatomical structure in the digestive tract, preferably the buccal cavity; the pharynx; the small intestine, including the duodenum, jejunum, and ileum; the stomach, including the esophagus, cardia, and pylorus; the cecum; the rectum ... The tissue may be selected from the large intestine, including the intestine, colon, rectum and anal canal, i.e., the imaged tissue may be subject to peristaltic movement during a medical procedure.

[0051] Alternatively, the imaged tissue may be part of the skin of a subject, for example, the imaged tissue is part of a wound of a subject.

[0052] system As previously described, the present disclosure further relates to a system for identifying blood vessels in tissue during a medical procedure on a subject, the system comprising: continuously generating fluorescent signals from blood vessels in the tissue, the intensity of the fluorescent signals oscillating in a predetermined pattern; continuously receiving fluorescent images of the tissue; and analyzing the fluorescence images and their associated vibration intensities, thereby sequentially identifying blood vessels in the tissue; It is configured for:

[0053] In a preferred embodiment, the system further comprises a controllable injection pump for holding at least one first fluorescent imaging agent, the injection pump configured to inject a series of defined boluses of the first fluorescent imaging agent into a vein of the subject, thereby generating a fluorescent signal oscillating in the predetermined pattern.

[0054] The fluorescence image may be received by a suitable imaging unit (e.g., a camera, e.g., a surgical, laparoscopic, or microscopic camera (e.g., a video camera)) that may be part of the system of the present disclosure. The analysis may be provided by a processing device, either locally or as part of a cloud service.

[0055] The systems of the present disclosure may be configured to perform all of the steps of the methods of the present disclosure.

[0056] The prescribed bolus preferably corresponds to less than 0.01 mg ICG / kg body weight of the first fluorescent imaging agent. The prescribed bolus may also correspond to less than 0.5 mg ICG of the first fluorescent imaging agent. Preferably, the fluorescent agent is ICG, and the amount of ICG in the prescribed bolus is preferably less than 0.01 mg / kg body weight of the subject. Thus, preferably, the amount of ICG in the prescribed bolus is less than 1 mg ICG or less than 0.5 mg ICG.

[0057] The system may be configured to inject boluses at intervals of between 5 and 600 seconds (eg, between 15 and 300 seconds, eg, between 45 and 210 seconds, eg, between 90 and 120 seconds).

[0058] A further advantage of the disclosed approach is the opportunity to identify local networks of blood vessels: by briefly clamping a freely visible vessel, its associated perfusion region is delimited, and by subsequently observing its fluctuating ICG signal, its associated network becomes clearly visible because perfusion in that region changes rapidly.

[0059] Intraoperative fluorescence imaging Perfusion (e.g., blood flow) can be imaged intraoperatively and assessed in real time using near-infrared light from a surgical microscope or camera to acquire video images of fluorescence in the near-infrared region excited by a fluorescent angiographic agent administered intravenously as a tracer. The state of perfusion can thereby be ascertained in real time. In this disclosure, perfusion in blood vessels is used to identify blood vessels using fluorescence imaging, but is not necessarily limited to intraoperative use of a surgical camera.

[0060] The systems and methods of the present disclosure may provide enhanced information of tissue properties, including the location of superficial and deeper blood vessels, particularly when different fluorescent agents are used, because careful selection of different fluorescent agents provides the option of obtaining perfusion information from different depths in the tissue.

[0061] During medical procedures involving fluorescence imaging, such as diagnostic, screening, examination, and / or surgical procedures, a solution containing a fluorescent contrast agent (e.g., ICG) is intravenously injected, and the molecules are excited by an infrared source (e.g., a laser with a wavelength in the infrared wavelength range (e.g., approximately 780 nm)). Fluorescence light with a wavelength of approximately 830 nm is then emitted from the excited contrast agent molecules and can be recorded using an imaging device, such as a camera. A filter can be provided to block the excitation light, since the excitation intensity is typically much greater than the fluorescence intensity. The excitation intensity can be approximately 1 W per emission angle, while the fluorescent power per pixel can be approximately 0.15 pW. Despite the difference of several orders of magnitude, a good signal-to-noise ratio (SNR) can be achieved. The recorded fluorescence provides an image of perfusion in the imaged tissue, allowing deeper vessels to be seen due to the 5-10 mm penetration depth of ICG. Because ICG molecules bind to proteins in the blood, the video images contain information about the level of perfusion—information that can be difficult for surgeons to quantify intraoperatively if only the acquired video images are available.

[0062] In the systems and methods of the present disclosure, the fluorescent contrast agent is selected from the group of indocyanine green (ICG) and fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalaldehyde, fluorescamine, rose bengal, trypan blue, fluorogold, green fluorescent protein, flavin, methylene blue, porphysome, cyanine dye, IRDDye800CW, CLR 1502 in combination with a targeting ligand, OTL38 in combination with a targeting ligand, or combinations thereof.

[0063] Indocyanine green (ICG) is a cyanine dye used in medical diagnostics and is by far the most common dye for perfusion assessment. It has a peak spectral absorption at approximately 800 nm. These infrared frequencies penetrate the retinal layer, allowing ICG angiography to image deeper circulation patterns than fluorescein angiography. ICG binds tightly to plasma proteins and is confined to the vasculature. It is administered intravenously and is excreted from the body by the liver into the bile with a half-life of approximately 3-4 minutes, depending on liver function. ICG sodium salt is usually available in powder form and can be dissolved in various solvents; 5% (<5% depending on the batch) sodium iodide is usually added to ensure better solubility. Sterile lyophilized water-ICG solutions are approved in many European countries and the United States as diagnostic agents for intravenous use under the names ICG-Pulsion, IC-Green, and VERDYE.

[0064] The absorption and fluorescence spectra of ICG are in the near-infrared region. Typically, a laser with a wavelength of approximately 780 nm is used for excitation. At this wavelength, the fluorescence of ICG can be detected by filtering out the scattered light from the excitation light.

[0065] Although the toxicity of ICG is classified as low, administration is not without risk (e.g., during pregnancy). It is known that ICG decomposes into toxic waste products under the influence of UV light, producing many as-yet-unknown substances. Thus, it is in the patient's best interest that the dose of ICG used during fluorescence imaging be minimized as shown herein.

[0066] Fluorescein is another dye that is widely used as a fluorescent tracer for many applications. Fluorescein has an absorption maximum of 494 nm and an emission maximum of 512 nm (in water). Therefore, it is suitable for use in combination with ICG because the absorption and emission wavelengths of the two dyes are separated by several hundred nanometers.

[0067] Dosage regimen The present disclosure further relates to a method for automated perfusion assessment of a subject's anatomical structure, comprising administering intravenously a bolus of approximately 1 / 10 the usual dose used for perfusion assessment. For indocyanine green (ICG), the usual bolus is 0.1-0.3 mg / kg body weight. According to the present disclosure, a bolus of less than 0.01 mg / kg body weight, preferably less than 0.005 mg / kg body weight, more preferably less than 0.0049 mg / kg body weight of a first fluorescent imaging agent such as ICG may be used, even more preferably less than 0.0048 mg / kg body weight, even more preferably less than 0.0047 mg / kg body weight, most preferably less than 0.0046 mg / kg body weight, and even more preferably less than 0.004 mg / kg body weight of the first fluorescent imaging agent. For the other fluorescent imaging agents described herein, the bolus may be similarly reduced according to the present disclosure. As mentioned above, the agent can be injected by a controllable injection pump, for example, as a series of boluses with a defined time between subsequent boluses. After injection of each bolus, the fluorescence emission from the anatomical structure can be measured. This method can be combined with the methods of the present disclosure for automatic perfusion assessment of a subject's anatomical structure, particularly for bolus administration regimens, to sequentially identify blood vessels in tissue.

[0068] The smallest bolus that provides a quantifiable fluorescence emission representative of perfusion of an anatomical structure and / or identifiable blood vessels can be determined after administering a series of increasing boluses, which may contain gradually increasing or decreasing amounts of agent, for example, the amount may increase or decrease in 10% increments from one bolus to a subsequent bolus.

[0069] The boluses are preferably provided as a regular series of injections with each bolus at a defined and regular time interval. The interval between boluses can be between 5 and 600 seconds, e.g., between 5 and 300 seconds, e.g., between 10 and 180 seconds, e.g., between 10 and 140 seconds, e.g., between 10 and 90 seconds, e.g., between 15 and 80 seconds, e.g., between 20 and 70 seconds, e.g., between 30 and 60 seconds. In another embodiment, the interval between boluses can be between 5 and 600 seconds, e.g., between 10 and 600 seconds, e.g., between 15 and 600 seconds, e.g., between 15 and 300 seconds, e.g., between 30 and 240 seconds, e.g., between 45 and 240 seconds, e.g., between 90 and 240 seconds, e.g., between 90 and 120 seconds. Preferably, the interval between boluses can be between 60 and 600 seconds, or between 120 and 600 seconds. The interval between the boluses is preferably long enough to allow measurement of the perfusion slope for each bolus in the anatomy, preferably where the perfusion slope includes slope onset and washout slope.

[0070] For ICG, the amount of the fluorescent imaging agent is preferably between 0.0001 and 0.001 mg / kg body weight / bolus, for example, between 0.001 and 0.01 mg / kg body weight / bolus, preferably between 0.0005 and 0.005 mg / kg body weight / bolus, more preferably between 0.001 and 0.004 mg / kg body weight / bolus. The initial amount is advantageously at least 0.001 mg / kg body weight, preferably less than 0.005 mg / kg body weight. Subsequent boluses may then increase from one bolus to the next by at least 0.001 mg / kg body weight, preferably less than 0.005 mg / kg body weight / bolus. For other types of fluorescent imaging agents, the dose is preferably selected based on their fluorescence relative to ICG. Thus, fluorescent imaging agents with higher emission rates are preferably administered at correspondingly lower doses. The dose may, for example, be substantially inversely linear with the quantum yield of the fluorescent imaging agent. The dose may further be based on the absorption and emission spectra relative to ICG.

[0071] The bolus preferably has a liquid volume of 0.5 μL to 10 mL, for example, 0.5 to 5 mL. That is, the amount of the first fluorescent imaging agent is preferably dissolved in the liquid. In a preferred embodiment of the present disclosure, a certain volume of isotonic solution (e.g., saline) is injected immediately after the injection of the bolus of fluorescent imaging agent. For example, the volume of the isotonic solution here is 1 to 20 mL, for example, 2.5 to 15 mL, for example, 5 to 10 mL.

[0072] In a further embodiment of the present disclosure, a second fluorescent imaging agent is administered, said second fluorescent imaging agent having an emission maximum that differs from the emission maximum of the first fluorescent imaging agent by at least 50 nm, or at least 100 nm. The first and second fluorescent imaging agents are preferably administered alternately. Advantageously, the interval between administrations of different fluorescent imaging agents is half the interval between subsequent administrations of the same fluorescent imaging agent.

[0073] In a further embodiment of the disclosed method, a series of fluorescent images of an anatomical structure and / or tissue is generated for assessment of perfusion and / or identification of blood vessels. The fluorescence may be automatically detected by illuminating the anatomical structure / tissue with a light source capable of exciting a fluorescent imaging agent, and the emitted light is quantified and / or analyzed through the series of fluorescent images of the anatomical structure / tissue.

[0074] The period between boluses can be determined by a computer configured to detect the perfusion gradient caused by each bolus. Furthermore, the amount of fluorescent imaging agent in the bolus can be controlled by a computer configured to determine the minimum bolus corresponding to the minimum fluorescence emission representative of perfusion of the anatomical structure. This computer can be part of the system of the present disclosure.

[0075] In further embodiments, the perfusion assessment includes locating perfusion complications in an anatomical structure. Thus, the perfusion assessment can be used in connection with diagnostic or surgical procedures. For example, the procedures include laparoscopic diagnostic examinations, laparoscopic exploration, laparoscopic surgery involving traditional laparoscopy, robotic surgery, and open surgery. The procedure can alternatively include the creation of an anastomosis (e.g., intestinal anastomosis, wound, plastic surgery, cardia surgery, or cancer).

[0076] Further embodiments of the present disclosure relate to fluorescent imaging agents for use in the methods disclosed herein. Still further embodiments relate to the use of fluorescent imaging agents in the preparation of medicaments for use in automated vascular perfusion assessment and / or sequential identification methods as disclosed herein.

[0077] In a further embodiment of the present disclosure, the fluorescent imaging agent is injected repeatedly. In certain cases, a longer period may be required, for example, at least 2 minutes, preferably at least 3 minutes, even more preferably at least 4 minutes, even more preferably at least 5 minutes, most preferably at least 8 minutes, and most preferably at least 10 minutes. In this case, the fluorescent imaging agent is injected repeatedly. Therefore, the background level is reduced to an acceptable level (e.g., below a certain percentage of maximum fluorescence intensity) or until no substantial fluorescence can be measured, and injection of the fluorescent imaging agent can be continued.

[0078] A system of the present disclosure may be configured to perform the method of the present disclosure, which may be provided by a system having at least one processor and a memory having instructions stored thereon, which when executed by one or more processors cause the system to perform the method of the present disclosure for automated perfusion assessment of an anatomical structure.

[0079] Vibration Dynamics The inventors further realized that measurement and analysis of repeatable bolus injections can be further extended from interpretation and quantification of single inflow and / or single outflow phases to analysis of oscillating fluorescence dynamics, which can reveal physical perfusion characteristics previously unavailable without invasive measurements.

[0080] The disclosed systems and methods can be configured for repeated injections of small boluses (e.g., minimal boluses) at regular intervals. These boluses can result in periodic variations that, when measured, take the approximate form of a sinusoidal curve, depending, for example, on the injection time interval. In such a curve, the measured intensity signal is expected to increase with the influx of fluorescent imaging agent from a given bolus, then decrease during the washout phase of the bolus, until increasing again with a subsequent bolus, etc., resulting in a periodic (sinusoidal) pattern.

[0081] Preferably, the disclosed system can be configured to recognize parameters (e.g., frequency and / or amplitude) of the vibration intensity curve. The trained system can then predict both the directionality and regularity of the upcoming signal dynamics. The system preferably uses measured values ​​to recognize vibration patterns, so that the system can then detect discrepancies between measured and predicted values. The measured values ​​can be further used continuously to improve pattern recognition, i.e., predicted values. Alternatively or additionally, injection parameters (e.g., bolus frequency, dose, and flow rate) can be used to determine the predicted value (i.e., vibration pattern).

[0082] Once the system anticipates that predicted value, it can detect and alert to the onset of an ischemic condition at an early point—ideally immediately. Detection of an ischemic condition can be a function of the predicted value and its detected value (e.g., a threshold value). However, with the disclosed approach of sequentially identifying vessels, an ischemic condition, or any type of disruption or breach in a vessel or vascular network, can be visually observed almost immediately by appropriate medical personnel.

[0083] Deviations from the expected sinusoidal pattern can be caused, for example, by the onset of an ischemic condition in at least a portion of the anatomical structure / tissue visible in the video image, or by local changes in perfusion to the indicated region. An illustration showing this change in dynamics due to the onset of ischemia in a human subject is shown in FIG. 2A, and a closer zoom is shown in FIG. 2B. As can be seen, it is possible to detect a transition from a regular oscillating fluorescent signal to an ischemic plateau. However, it should be noted that changes to the perfusion of the anatomical structure of interest can result in other measured patterns in addition to an ischemic plateau. One example is venous occlusion, where the outflow of blood from the anatomical region is blocked or reduced, resulting in the concentration or pooling of fluorescent agent in the indicated region. This results in a change in the vibration dynamics due to the periodic oscillations. As can be seen in Figure 4, the result is not a plateau during the periodic oscillations.

[0084] Such a system, as described herein, can observe and detect changes in perfusion levels in a depicted region in a video image within seconds. This can be detected in regions observed for extended periods (e.g., minutes), where the dynamics are continuously visualized and, therefore, their phases are well known. An illustration highlighting the signal differences one might expect to observe for ischemic / healthy tissue regions is shown in Figure 2C. However, it can equally be determined in anatomical regions visualized only for short time intervals (e.g., 10-20 seconds), because the described system is trained to predict and detect a specific phase of the described oscillating dynamic signal at the depicted time in tissue, consisting of regular rises and falls in the time-intensity signal. See Figure 2D, which illustrates what an anatomical region of interest might look like if it drifts in and out of focus of the recorded image.

[0085] Preferably, the system includes tracking means and can run independently in the background, while the surgeon is only exposed to the visible white light signal and is therefore only interrupted / informed by a warning signal, e.g., during detection of the onset of an ischemic condition.

[0086] Another aspect of the present disclosure relates to continuous perfusion assessment associated with repeated injections of a fluorescently active agent and monitoring the resulting oscillation curve. In addition to detecting unexpected changes in perfusion, the system can be used to assess the perfusion area of ​​an artery. As an example, a surgeon may consider cutting an artery as part of a surgical procedure. Before cutting the artery, the surgeon may temporarily restrict perfusion through the artery, and the disclosed approach may allow visualization of the artery's perfusion area within a short period of time (e.g., less than one minute). This may be valuable information for the surgeon during subsequent surgical procedures. In a similar manner, the system can be used to assess the drainage area of ​​a vein or a group of veins, lymphatic vessels, lymph nodes, or other portions of the circulatory and / or lymphatic pathways. By temporarily restricting blood flow through a blood vessel, blood pools in the anatomical area normally drained by that vessel or group of vessels. This allows visualization of the anatomical area drained by the vessel within a relatively short period of time (e.g., less than two minutes). This can provide important information to surgeons in fields such as general surgery and plastic surgery (including wound and reconstructive surgery), for example, during subsequent surgical procedures.

[0087] digestive tract Complications involving the gastrointestinal tract are often related to local hemodynamics. That is, changes in normal hemodynamic status can be an indicator of an increased risk of complications. Perfusion assessment of the gastrointestinal tract, particularly within and near the surface of the gastrointestinal tract, such as the tissues of the gastrointestinal wall, can therefore be an important diagnostic tool when examining the gastrointestinal tract, for example, during laparoscopic diagnostic examinations, laparoscopic surgery involving laparoscopy or traditional laparoscopy, or robotic surgery, and open surgery, for example, to diagnose or locate complications. Perfusion assessment is also important during the surgical procedure of creating an anastomosis, which can be provided to establish communication between two previously separate portions of the gastrointestinal tract. As an example, an intestinal anastomosis establishes communication between two previously separate portions of the intestine, typically restoring intestinal continuity after removal of a pathological condition affecting the intestine. Intestinal anastomoses may be provided, for example, for 1) restoration of intestinal (e.g., bowel) continuity after resection of pathological bowel, and 2) bypass of unresectable pathological intestine (e.g., bowel). Certain pediatric conditions may also require intestinal anastomoses.[6]

[0088] Resection of pathological bowel may be performed in the following situations: Intestinal gangrene due to vascular compromise caused by mesenteric vascular disease, prolonged intestinal obstruction, intussusception, or volvulus Malignant tumors Benign conditions (e.g., intestinal polyps, intussusception, roundworm infection associated with intestinal obstruction) Infections (e.g., tuberculosis complicated by stricture or perforation) ·Traumatic perforation Large perforations (traumatic) that are not amenable to primary closure Radiation enteritis complicated by bleeding, stricture, or perforation, Inflammatory bowel disease, ulcerative colitis, or Crohn's disease that is refractory to medical treatment or associated with complications (e.g., bleeding, perforation, toxic megacolon, dysplasia / carcinoma) Chronic constipation, idiopathic slow transit constipation, or Hirschsprung's disease: If the above conditions are refractory to medical treatment, a subtotal colectomy may be performed.

[0089] Bypass of unresectable pathological bowel may be performed in the following situations: Locally advanced tumors causing luminal obstruction Metastatic disease causing bowel obstruction Poor general condition or conditions that prevent major resection

[0090] Pediatric conditions that may require an intestinal anastomosis include: Congenital anomalies (e.g., Meckel's diverticulum, intestinal atresia, malrotation with intestinal volvulus leading to gangrene, meconium ileus, gastric duplication cyst, Hirschsprung's disease) Inflammatory conditions (e.g., necrotizing enterocolitis, enteritis, tuberculosis, intestinal perforation) Other conditions (e.g., intussusception, angiodysplasia, polypoid disease, ascariasis) As part of other surgical procedures (e.g., Kasai portoenterostomy, choledochal cyst, urinary diversion, pancreatic tumors)

[0091] Postoperative complications associated with anastomoses in the digestive tract are unfortunately frequent and often result from insufficient perfusion (capillary blood supply) at the anastomosis (i.e., the joining of two sections of the tract). Inadequate perfusion can lead to anastomotic leakage, a serious and frequent complication associated with, for example, colon surgery (more than 10% of the procedures result in complications). Within colon cancer surgery, more than 30% of patients with anastomotic failure die from postoperative complications, and approximately 25% of the remaining patients will suffer from a stoma for the rest of their lives. Risk factors associated with anastomotic failure include anastomotic tension, tissue damage, and, in particular, reduced blood perfusion.

[0092] The present disclosure, therefore, according to one embodiment, relates to performing image analysis of one or more video sequences representing at least a portion of the gastrointestinal tract, acquired before, during, and / or after surgery, particularly surgery involving the gastrointestinal tract. This may be particularly applicable to gastrointestinal surgery - thus, the video sequences may preferably include at least some outer portion of the gastrointestinal tract, such that perfusion in at least a portion of the gastrointestinal tract wall may be measured and assessed.

[0093] The digestive tract is the organ system in humans and other animals that ingests food, digests it to extract and absorb energy and nutrients, and excretes remaining waste products as feces and urine. The digestive tract may be understood as the tube that transports food to the digestive system. Thus, the term digestive tract, as used herein, includes the buccal cavity; the pharynx; the small intestine, including the duodenum, jejunum, and ileum; the alimentary canal ...alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary canal; the alimentary can the stomach, including the esophagus, cardia, and pylorus; and the large intestine, including the cecum, colon, rectum, and anal canal.

[0094] clinical application Visualization of vascular anatomy as disclosed herein is of great importance during almost any type of surgery, as continuous detection of blood vessels reduces the risk of unintentional severance of the vessel. The systems and methods of the present disclosure may also be used, inter alia, within the following clinical applications:

[0095] Abdominal / General surgery Excision surgery, to quickly identify the exact location of the vessel to be cut / ligated. Ischemic bowel surgery, to quickly determine which and where blood vessels are blocked and anatomical structures are poorly perfused. Acute abdominal pain, to identify the underlying pathology, e.g., to help rule out ischemia. Repeat surgery, in previously operated patients with numerous surgical adhesions. To detect blood vessels in cancer surgery, such as in resection surgery, performed by general surgeons. General surgery, involving virtually any organ in the abdominal tract, for example, performing an anastomosis or surgery on the stomach (ventricle). General surgery, involving superficial or deep infections, to detect and map blood vessels.

[0096] thyroid surgery Thyroid surgery, including the removal of thyroid tissue. Continuous detection of blood vessels is a significant advantage because thyroid surgery carries the risk of excessive bleeding. Thyroid surgery also carries the risk of ablation or removal of one or more portions of the parathyroid glands, and the disclosed approach can be applied to identify and map blood vessels in the relevant area so that the parathyroid glands are more easily visible to the surgeon.

[0097] Pelvic surgery Gynecological / urological surgery, to quickly locate the exact vessels to be cut / ligated, and to identify and identify vessels intraoperatively. Cancer surgery, to detect blood vessels.

[0098] plastic surgery Skin grafting, to rapidly identify the exact location of the donor vessel to be cut / ligated and to monitor the perfusion of the same vessel on the recipient; in the acute situation during surgery, and also in the days that follow, to monitor healing and angiogenesis. In all skin areas near a surgical procedure, to detect and map vascular structures in an anatomical region before the procedure begins, and during the procedure if the surgeon deems this necessary for further information. That is, the disclosed approach can be used as a clinical tool to identify and map a patient's blood vessels and their associated vascular anatomy days or weeks before a surgical procedure, allowing medical personnel time to carefully plan the procedure. This mapping can be performed, for example, according to the disclosed approach, by measuring fluorescent signals through the skin of the area of ​​interest over a 30-45 minute period and creating a 2D or 3D map for medical personnel to use in planning. The identified vascular map can be integrated with other tests (e.g., CT scans, MR scans, or ultrasound scans). The disclosed approach has the advantage of enabling the identification and mapping of blood vessels that are too small to be accurately mapped in a CT scan, for example.

[0099] EAT THROAT AND NECK SURGERY Various procedures including: facial cosmetic surgery, tracheotomy, cancer, etc.

[0100] orthopedic surgery Amputation, to rapidly identify and map blood vessels before a limb or anatomical area is amputated, to select the exact and best site for amputation, to reclose the skin and ensure optimal healing. To detect and map blood vessels in areas where debridement or similar procedures are required, such as for infections.

[0101] cardia surgery To rapidly identify and locate peripheral vessels to the heart during CABG, bypass surgery.

[0102] vascular surgery Amputation surgery, to quickly identify and locate the exact vessels to be cut / ligated as described above. Vessel harvesting for bypass surgery, to quickly identify and locate the exact vessel to be cut / ligated. [Example]

[0103] Example The intensity curves shown in Figures 1A-B are the result of bolus injections of a regular amount of fluorescent agent (in these cases, ICG). The amount of ICG in each bolus was selected so that the fluorescent emission was visible to the human eye. Examples are provided to illustrate various perfusion parameters that can be calculated after fluorescence imaging. To a large extent, these same parameters can also be determined after injection of much smaller doses (i.e., the micro-dose approach disclosed herein, possibly with repeated and serial measurements of perfusion and associated assessment and vascular identification).

[0104] Figure 1A shows an example of an intensity curve acquired from tissue, e.g., from a region of interest in a video sequence, after a bolus of ICG has been administered to a subject. Similar data could be obtained using a different contrast agent. The intensity is essentially zero until a sudden increase in intensity indicates the passage of ICG molecules in the imaged tissue, which are excited to fluoresce. The intensity peak is followed by a gradual washout of the ICG molecules. Intensity is shown in arbitrary units.

[0105] 1B is the corresponding intensity curve, where the hemodynamic parameters perfusion slope, slope start, slope end, maximum intensity, washout slope, washout start, and washout slope end are calculated and plotted. Assessment of perfusion parameters is further disclosed in pending application WO 2018 / 104552, which is incorporated herein by reference in its entirety.

[0106] Figure 2A shows actual measurement data from a human subject who was repeatedly injected with microboluses of ICG at regular intervals (in this example, the intervals were approximately 2 minutes). The first microbolus of ICG contained an amount of 0.00456 mg ICG / kg human subject body weight, and each subsequent microbolus of ICG contained an amount of 0.00456 mg ICG / kg human subject body weight. The same amount of ICG / kg of human subject body weight was included. The time-intensity curve shows a substantially sinusoidal pattern with a linear increase over time. The increase in intensity over time is related to the ratio between the fluorescent agent dose and the washout time (during which the fluorescent intensity decreases). At a certain point (approximately t = 3800 s, Figure 2B), perfusion is restricted, causing the onset of ischemia. This After this point, a lack of oscillation can be seen, forming what can be described as an ischemic plateau.

[0107] FIG. 2C shows idealized data showing a sinusoidal time-intensity curve. The measured ROI intensity increases upon injection of the fluorescent imaging agent and decreases during the washout phase. At approximately t=3750 seconds, the measured data shows a constant measured ROI intensity value due to the onset of ischemia. Alternatively, if there was no ischemia, the measured value would instead be expected to follow a dashed line, such that the measured ROI value continuously follows a sinusoidal pattern.

[0108] Figure 2D shows idealized data showing sinusoidal time-intensity curves without ischemia as the anatomical region of interest drifts in and out of focus. The dashed line indicates the measurements that would be expected if the ROI were continuously observable. If this is not possible, for example, due to the anatomical region of interest drifting in and out of focus in the recorded image, the measured data may not be complete, but instead may have gaps—time intervals during which no measurement data was acquired for the anatomical region of interest. Thus, the system described above: Preferably, the sinusoidal pattern can be recognized even when the recorded data is incomplete because the phase of the dynamics is known. If the system can accurately recognize the sinusoidal pattern, it is provided along with a predicted intensity value of the ROI at each time point, which can then be used for comparison with the measured value. If the measured value differs from the expected value, the system can be configured to provide a warning to the surgeon. Thus, the system can be configured to recognize the phase of the oscillating / sinusoidal pattern at a measured time point or interval, which is then compared with the predicted phase for that time point or interval, where the predicted phase is preferably based on the recognized oscillation pattern or / and the known frequency of repeated bolus injections. As a result, the system does not necessarily require consecutive measurements, but instead can be based on the predicted phase of the oscillation pattern in combination with the time information of the measured time point or interval, such that a specific phase of the oscillation pattern is predicted to be present in the measured interval.

[0109] Figure 3A shows fluorescence intensity measurements of a human subject taken over a longer time interval (approximately 40 minutes), in which the human subject was repeatedly injected with microboluses of ICG. The first microbolus of ICG contained 0.006 mg ICG / kg human subject body weight, and each subsequent microbolus contained the same amount of ICG at 0.006 mg ICG / kg human subject body weight. The measured intensities of seven distinct ROIs were assigned distinct colors in the graph. The measured fluorescence intensity exhibited a periodic sinusoidal pattern, the frequency of which corresponded to the injection frequency (approximately 120 s). The pattern increased substantially linearly due to the accumulation of the fluorescent imaging agent, due to the relatively short period of injection compared to the dose size. At approximately t = 2000 s, the repeated injections of the fluorescent imaging agent were stopped, causing a near-exponential decay of the fluorescence intensity.

[0110] Figure 3B shows an enlargement of the marked area in Figure 3A. Here, smaller variations can be seen within the same ROI and between different ROIs. At the same time, the periodic intensity pattern is distinct from the pattern of each ROI with the same period.

[0111] Figure 4 shows a time-intensity plot of measurements made in a human subject by repeated injection of microboluses of a fluorescent imaging agent. The first microbolus of ICG contained an amount of 0.00456 mg ICG / kg human subject body weight, and each subsequent microbolus of ICG contained the same amount of 0.00456 mg ICG / kg human subject body weight. The graph shows the results of venous occlusion, where perfusion is limited, but not completely prevented, between approximately t = 62 and 78 minutes. In this case, the oscillatory dynamics of the measured fluorescence intensity ceased. Thus, the measurements show an irregular increase during venous occlusion. It should be noted, therefore, that reduced perfusion does not necessarily result in a plateau, as typically occurs during ischemic conditions.

[0112] Figure 5A shows a snapshot of the ICG analysis tool running on a humanoid subject. The acquired images in Figures 5-10 show a site on the right forearm, where fluorescent signals are visible across the skin of the arm. The image in Figure 5A was taken very early in the bolus injection, where the ICG microbolus has just been injected and is beginning to enter the artery. Some arteries can be identified. The four boxes in the image indicate measurement regions (also known as regions of interest (ROIs)). Four ICG intensity curves are shown on the right (one for each POI). One of the ROIs is located in an artery, and its corresponding intensity curve is the highest. One ROI is located in a tissue region, and its associated intensity curve indicates that some ICG has already diffused into the tissue. Two ROIs are located in veins and are nearly flat, indicating that ICG has not yet left the tissue region and been transported back via the veins. The intensity curves of the two venous ROIs coincide and cannot be distinguished from each other. The phase differences of the four ICG intensity curves are clearly visible, so if the expected phases of the arteries, veins, and tissues are known, the four ROIs in Figure 5A can be classified as arteries, veins, and surrounding tissues, respectively.

[0113] Figure 5B shows a snapshot several seconds later than Figure 5A, in which multiple arteries can be visually identified. Some dark areas are still present where the ICG has not yet spread; that is, the ICG is still entering the subject. All four ICG intensity curves for the four ROIs steadily increase. However, as can be seen from the curves, there is a clear phase difference for three groups of curves: 1) artery, 2) tissue, and 3) vein. The artery ROI is in front of the other two groups. The tissue ROI is "behind" the artery curve, and the tissue ROI is "in front" of the vein ROI. Note that the phase difference in both directions is more or less equal; that is, the tissue is approximately halfway through the ICG molecule's journey from artery to tissue to vein. Again, an ROI can be classified as either an artery, vein, surrounding tissue, or other if its expected phase is known. This can be provided for all pixels in the image or for groups of pixels. That is, every pixel can be classified as an artery, vein, surrounding tissue, or other if its expected phase is known. That is, image filtering can provide a visual identification of blood vessels, but if the phases of the various signals are known for a given vibration pattern, all or most of the pixels in the image can be classified; much more detailed information is obtained. This can be utilized, for example, when overlaying blood vessels onto white-light images, and also when it is needed to track objects in a sequence of images where movement, e.g., peristaltic movement, is occurring.

[0114] Figure 6A shows a snapshot taken a few seconds later than Figure 5B, when the fluorescence intensity from a particular microbolus of ICG is reaching its peak value. After this point, more ICG begins to leave the tissue region than enters it. Comparing this image with the snapshots shown earlier in Figures 5A-B, it can be seen that almost all regions are visible here. The darkest regions are now veins, which have not yet begun to transport ICG away from the tissue. This is also reflected in the corresponding ICG intensity curves on the right. The ICG curves are all still increasing. That is, we are still in the arterial-dominated phase, but the concave shape of the curves indicates that peak intensity is approaching. However, a phase difference is still discernible between the ICG intensity curves. In this snapshot, blood vessels are not clearly identifiable because too much ICG has entered the tissue region from the arteries and not enough ICG has yet entered the veins. However, veins may actually be identifiable as dark regions.

[0115] Figure 6B shows a snapshot approximately one minute later than Figure 6A, illustrating the washout phase in which veins can be clearly identified. The corresponding perfusion analysis from the associated ICG curves also shows that the venous ROI clearly has the highest intensity, but the time scale is different in the washout phase. That is, it may require a longer time scale to evaluate the phase difference between the artery, tissue, and vein in the washout phase. However, the phase difference is visible in the ICG curves.

[0116] FIG. 7 shows an edge-filtered version of FIG. 6B illustrating one example of visual enhancement that can be provided to medical personnel during a medical procedure by utilizing the approach of the present disclosure. Once the phase difference is known for a particular situation, it is known when arteries and veins are optimally identified, respectively. In FIG. 7, the veins are visually enhanced, allowing the surgeon to avoid accidentally cutting any veins. A pixel-by-pixel approach is used in FIG. 7 in combination with edge filtering, followed by smoothing filtering, as with FIG. 6B. The result is a visibly darkened image overall, except for the major veins. This information can be overlaid on any screen visible to the surgeon during surgery (even white-light image screens) to improve the basis on which the surgeon draws his or her decisions.

[0117] Figure 8 shows an image in which arteries have been identified in the sequence of images shown in Figures 5A-6B. The arteries are visually enhanced in black, as can be seen in the grayscale image.

[0118] Figure 9 shows an image in which veins have been identified in the sequence of images shown in Figures 5A-6B. The veins are visually enhanced in black, as can be seen in the grayscale image.

[0119] Figure 10 shows an image in which the arteries and veins shown in Figures 8-9 are visually enhanced in red (arteries) and blue (veins) and superimposed in an image so that the arteries and veins are clearly visible and easily identifiable. This is an example of an augmented reality (AR) view that may be provided to a surgeon during a medical procedure using the approach of the present disclosure. Red pixels are mapped to arterial groups, and blue pixels are mapped to venous groups. These mappings may be continuously updated as a microbolus regimen may be performed in the background. In practice, a given group (e.g., arterial group) has a known phase that may be identified during the initial learning phase of the microbolus regimen (where the arterial phase may be associated with the time of each bolus injection). Once the arterial phase is known, a segment of the fluorescent signal (e.g., a few seconds) is sufficient to assess the associated perfusion of one or more pixels or ROIs in the image sequence and calculate a phase matching score. If the phase at a pixel / ROI matches the arterial phase, the phase matching score is high, and the pixel can be identified as an artery and colored red. A similar cycle can be performed for veins. Such phase matching assessment can be provided during both the inflow and washout periods of ICG. The disclosed approach to continuously identify blood vessels in tissue can therefore be performed continuously in the background.

[0120] item 1. A computer-implemented method for identifying blood vessels in tissue of a subject (during a medical procedure), said method comprising: successively acquiring fluorescence images of the tissue in which the fluorescence signal oscillates in a predetermined pattern; and analyzing said fluorescence images and their associated oscillating signals, thereby sequentially identifying blood vessels in said tissue; A method that encompasses

[0121] 2. The method of claim 1, wherein the identified vessels are combined to identify one or more networks of interconnected vessels.

[0122] 3. The method according to any of the preceding items, wherein the predetermined pattern is characterized by a frequency defined over a period of at least 10 minutes, or at least 15 minutes, or at least 30 minutes, or at least 1 hour, or at least 2 hours, with a period of between 30 seconds and 15 minutes, for example, between 1 and 10 minutes, or between 1 and 2 minutes, 2 and 3 minutes, 3 and 4 minutes, 4 and 5 minutes, 5 and 6 minutes, 6 and 8 minutes, or 8 and 10 minutes, and wherein the fluorescent signal oscillates with an intensity according to the predetermined pattern.

[0123] 4. The method according to any of the preceding items, wherein the identified blood vessels are continuously displayed and visualized on a screen.

[0124] 5. The method according to any of the preceding items, wherein the blood vessels are identified based on the phase difference between the fluorescent signal associated with the blood vessel and the surrounding tissue, respectively.

[0125] 6. The method of any of the preceding items, comprising generating at least one white light image of the tissue, wherein the identified blood vessels are visually enhanced.

[0126] 7. The method of any of the preceding items, wherein the white light images of the tissue are acquired sequentially.

[0127] 8. The method according to item 7, wherein the identified blood vessels are superimposed onto the white light image, visually enhanced, and displayed on a screen.

[0128] 9. A method according to any of the preceding items, further comprising determining a phase difference between a fluorescent signal originating from an artery in the tissue and a fluorescent signal originating from a vein in the tissue, and optionally a fluorescent signal originating from tissue surrounding the blood vessel, and relating this phase difference to the predetermined vibration pattern.

[0129] 10. A method according to any of the preceding items, wherein the sequence of acquired fluorescent images is analyzed and pixels in the fluorescent images are classified as either 1) arteries, 2) veins, 3) surrounding tissue, or 4) other based on the phase of the fluorescent signal at each pixel with respect to a predetermined vibration pattern.

[0130] 11. A method according to any of the preceding items, comprising identifying arteries and veins in the identified blood vessels based on a predetermined pattern of the oscillating fluorescent signal.

[0131] 12. The method according to item 11, wherein arteries and veins are distinguished based on phase differences in the associated fluorescent signals.

[0132] 13. The method according to any of items 11 to 12, wherein the identified arteries and veins are superimposed onto a white light image and visually enhanced so that the arteries and veins are visually distinguishable and displayed on a screen.

[0133] 14. The method according to any of the preceding items, wherein blood vessels are identified and / or visualized by image filtering (e.g., edge filtering).

[0134] 15. The method of any of the preceding items, wherein a series of boluses of at least one fluorescent imaging agent are provided into the subject's vein during a medical procedure, thereby generating a predetermined pattern of oscillating fluorescent intensity.

[0135] 16. The method of item 15, wherein the series of boluses are administered with a defined duration between successive boluses.

[0136] 17. The method of any of the preceding items, wherein the fluorescent imaging agent is ICG, and wherein each bolus of ICG corresponds to less than 0.01 mg ICG / kg body weight, preferably less than 0.005 mg ICG / kg body weight.

[0137] 18. The method of any of the preceding items, wherein the series of boluses are injected automatically by a controllable injection pump.

[0138] 19. The method of any of the preceding items, wherein the imaged tissue is part of an anatomical structure in the digestive tract, preferably selected from the buccal cavity; the pharynx; the small intestine, including the duodenum, jejunum, and ileum; the stomach, including the esophagus, cardia, and pylorus; and the large intestine, including the cecum, colon, rectum, and anal canal.

[0139] 20. The method of any of the preceding items, wherein the imaged tissue undergoes peristaltic movement during the medical procedure.

[0140] 21. The method of any of the preceding items, wherein the imaged tissue is part of an internal organ of the subject.

[0141] 22. The method of any of the preceding items, wherein the imaged tissue is a portion of the subject's skin.

[0142] 23. The method of any of the preceding items, wherein the imaged tissue is part of a wound in the subject.

[0143] 24. The method according to any of the preceding items, wherein the images are acquired during thyroid surgery, and blood vessels within one or more of the parathyroid glands are identified and made visible to medical personnel involved in the surgery.

[0144] 25. The method of any of the preceding items, wherein the at least one fluorescent imaging agent comprises indocyanine green (ICG), fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalaldehyde, fluorescamine, rose bengal, trypan blue, fluorogold, green fluorescent protein, flavin, methylene blue, porphysome, cyanine dye, IRDDye800CW, CLR 1502 in combination with a targeting ligand, OTL38 in combination with a targeting ligand, or a combination thereof.

[0145] 26. A system for identifying blood vessels in tissue during a medical procedure on a subject, wherein the system comprises: continuously generating fluorescent signals from blood vessels in the tissue, the fluorescent signals oscillating in intensity in a predetermined pattern; receiving sequential fluorescent images of the tissue; and The fluorescence image and its associated vibration intensity are analyzed to thereby identify blood vessels in the tissue. sequentially identifying The system is configured for.

[0146] 27. The system of item 26, comprising a controllable injection pump for holding at least one first fluorescent imaging agent, the injection pump configured to inject a series of defined boluses of the first fluorescent imaging agent into a vein of the subject, thereby generating a fluorescent signal oscillating in the predetermined pattern.

[0147] 28. The system of item 27, wherein the prescribed bolus corresponds to less than 0.01 mg ICG / kg body weight of the first fluorescent imaging agent.

[0148] 29. The system according to any of items 27 to 28, wherein the prescribed bolus corresponds to less than 0.5 mg ICG of the first fluorescent imaging agent.

[0149] 30. The system according to any one of items 27 to 29, wherein the fluorescent agent is ICG and the amount of ICG in the prescribed bolus is less than 0.01 mg / kg body weight.

[0150] 31. The system according to any one of items 27 to 30, wherein the fluorescent agent is ICG and the amount of ICG in the prescribed bolus is less than 1 mg ICG or less than 0.5 mg ICG.

[0151] 32. The system according to any of items 27 to 31, wherein the system is configured to inject boluses at intervals between 5 and 600 seconds, for example between 15 and 300 seconds, for example between 45 and 210 seconds, for example between 90 and 120 seconds.

[0152] 33. A method for automated perfusion assessment of an anatomical structure of a subject, the method comprising: intravenously administering a bolus equivalent to less than 0.01 mg ICG / kg body weight of a first fluorescent imaging agent; acquiring and analyzing time-series fluorescent images of tissue of the anatomical structure after injection of the first fluorescent imaging agent; and determining a perfusion parameter of at least one of the anatomical structures based on the analysis.

[0153] 34. The method of any of the preceding items, wherein the fluorescence emission from the anatomical structure is measured after injection of each bolus.

[0154] 35. A system for automated perfusion assessment of an anatomical structure during a medical procedure on a subject, the system comprising: a controllable injection pump for holding at least one first fluorescent imaging agent, the injection pump configured to inject a defined amount of the first fluorescent imaging agent into the blood of the subject, the system configured to receive and analyze time-series fluorescent images of tissue of the anatomical structure after injection of the first fluorescent imaging agent, and to determine at least one perfusion parameter of the anatomical structure based on the analysis.

Claims

1. For example, a computer-implemented method for identifying blood vessels in a tissue of a subject during a medical procedure, the method comprising: - successively acquiring fluorescent images of said tissue in which the fluorescent signal oscillates in a predetermined pattern having a constant frequency, said pattern being generated from a series of boluses of at least one fluorescent imaging agent, said series of boluses being administered with a defined and / or controlled duration between successive boluses that determines the frequency of said pattern; - analyzing at least a portion of said fluorescence image to determine: the time lag between the bolus injection and the arterial or venous fluorescent signal; - the time difference between the arterial and venous fluorescence signals; the time difference between the arterial or venous fluorescence signal and the tissue fluorescence signal; determining at least one time difference selected from the group consisting of: - sequentially identifying blood vessels in the fluorescence image of the tissue based on the time differences and the fluorescence signal oscillating in the predetermined pattern; A method that encompasses

2. 2. The method of claim 1, wherein the frequency of the pattern comprises a period between 1 and 5 minutes over a period of at least 15 minutes, and the fluorescent signal oscillates in intensity according to this predetermined pattern.

3. 10. A method according to any preceding claim, wherein the identified vessels are combined to identify one or more networks of interconnected vessels.

4. 10. The method according to any of the preceding claims, wherein the identified vessels are continuously displayed and visualized on a screen.

5. 10. The method of any of the preceding claims, comprising determining a time difference between an arterial fluorescence signal and a venous fluorescence signal, and identifying arteries and veins in the identified vessels based on the time difference and a predetermined pattern of the oscillating fluorescence signal.

6. 6. The method of claim 5, wherein the identified arteries and veins are superimposed onto a white light image and visually enhanced so that the arteries and veins are visually distinguishable and displayed on a screen.

7. 10. A method according to any of the preceding claims, further comprising analyzing the sequence of acquired fluorescence images and classifying pixels in the fluorescence images as either 1) arteries, 2) veins, 3) surrounding tissue, or 4) other based on the phase of the fluorescence signal at each pixel with respect to a predetermined oscillation pattern.

8. 10. The method according to any of the preceding claims, wherein blood vessels are identified and visualized by image filtering (e.g. edge filtering).

9. 10. The method of any of the preceding claims, wherein a series of boluses of at least one fluorescent imaging agent are provided into the subject's vein during image acquisition, thereby generating a predetermined pattern of oscillating fluorescent intensity, wherein the series of boluses are administered with a defined duration between successive boluses.

10. 10. The method of claim 9, wherein the fluorescent imaging agent is ICG, and wherein each bolus of ICG corresponds to less than 0.01 mg ICG / kg body weight.

11. 10. The method of claim 9, wherein the fluorescent imaging agent is ICG and each bolus of ICG corresponds to less than 0.005 mg ICG / kg body weight.

12. 10. The method of claim 9, wherein the fluorescent imaging agent is ICG and each bolus of ICG corresponds to less than 0.004 mg ICG / kg body weight, more preferably less than 0.003 mg ICG / kg body weight, even more preferably less than 0.002 mg ICG / kg body weight, and most preferably less than 0.001 mg ICG / kg body weight.

13. 10. The method of any of the preceding claims, wherein the series of boluses are injected automatically by a controllable injection pump.

14. 10. The method of any of the preceding claims, wherein the imaged tissue is part of an anatomical structure in the gastrointestinal tract, preferably selected from the buccal cavity; the pharynx; the small intestine, including the duodenum, jejunum, and ileum; the stomach, including the esophagus, cardia, and pylorus; and the large intestine, including the cecum, colon, rectum, and anal canal.

15. 10. The method of any of the preceding claims, wherein the imaged tissue undergoes peristaltic movement during the medical procedure.

16. 10. The method of any of the preceding claims, wherein the imaged tissue is part of an internal organ of the subject, or part of the skin of the subject, or part of a wound of the subject.

17. 10. The method of any of the preceding claims, wherein the images are acquired during thyroid surgery and blood vessels in one or more of the parathyroid glands are identified and made visible to medical personnel involved in the surgery.

18. 10. The method of any of the preceding claims, wherein the at least one fluorescent imaging agent is selected from the group of indocyanine green (ICG), fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalaldehyde, fluorescamine, rose bengal, trypan blue, fluorogold, green fluorescent protein, flavin, methylene blue, porphysome, cyanine dyes, IRDDye800CW, CLR 1502 in combination with a targeting ligand, OTL38 in combination with a targeting ligand, or a combination thereof.

19. A computer program having instructions that, when executed by a computing device or computing system, causes the computing device or computing system to perform a method for identifying blood vessels in tissue of a subject according to any of the preceding claims.

20. 1. A system for identifying blood vessels in tissue of a subject, the system comprising: - continuously generating fluorescent signals from blood vessels in said tissue, said fluorescent signals oscillating in a predetermined pattern having a constant frequency, said pattern being generated from a series of boluses of at least one fluorescent imaging agent, said series of boluses being administered with a defined and / or controlled duration between successive boluses that determines the frequency of said pattern; - receiving successive fluorescence images of said tissue, - analyzing at least a portion of said fluorescence image to determine: the time lag between the bolus injection and the arterial or venous fluorescent signal; - the time difference between the arterial and venous fluorescence signals; the time difference between the arterial or venous fluorescence signal and the tissue fluorescence signal; determining at least one time difference selected from the group - sequentially identifying blood vessels in the fluorescence image of the tissue based on the time differences and the fluorescence signal oscillating in the predetermined pattern; The system is configured for.

21. 21. The system of claim 20, comprising: a controllable injection pump for holding at least one first fluorescent imaging agent, the injection pump configured to inject a series of defined boluses of the first fluorescent imaging agent into a vein of the subject, thereby generating a fluorescent signal oscillating in the predetermined pattern.

22. 22. The system of claim 20, wherein the fluorescent agent is ICG, wherein the amount of ICG in the bolus is less than 0.005 mg / kg body weight, and wherein the system is configured to inject the bolus at intervals between 1 and 5 minutes.

23. A system according to any one of claims 20 to 22, wherein the system is calibrated to carry out the process according to any one of claims 1 to 18.

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