In vivo visualization of anatomic structures by fluorescein dyes

Abstract

The present invention relates to a method for visualizing an anatomic structure in an operating zone of a subject during a surgical procedure comprising (i) contacting said anatomic structure with a fluorescein dye; (ii) illuminating said operating zone with light exciting said fluorescein; and (iii) thereby visualizing said anatomic structure, wherein said anatomic structure is a nerve structure, and / or a fascia. The present invention further relates to methods, devices, systems nd kits related to the aforesaid method.

Description

[0001] Attorney Ref.: 097147-0181

[0002] DK17352PC

[0003] In vivo visualization of anatomic structures by fluorescein dyes

[0004] CROSS REFERENCE TO RELATED APPLICATION

[0005] The present application claims priority of US Provisional Application No. 63 / 833,844, filed on January 24, 2025, the contents of which is incorporated herein in its entirety.

[0006] BACKGROUND OF THE INVENTION

[0007] The present invention relates to a method for visualizing an anatomic structure in an operating zone of a subject during a surgical procedure comprising (i) contacting said anatomic structure with a fluorescein dye; (ii) illuminating said operating zone with light exciting said fluorescein dye; and (iii) thereby visualizing said anatomic structure, wherein said anatomic structure is a nerve structure, and / or a fascia. The present invention further relates to methods, devices, systems and kits related to the aforesaid method.

[0008] Nerve damage is a feared complication in surgical procedures, with over 94% of iatrogenic nerve injuries occurring during surgery1. Annually, 25 million patients are affected by this and over 100 million patients are at risk. Injuries to nerves can lead to chronic pain, motor and sensory deficits, or permanent loss of function, profoundly impacting quality of life. For example, surgeries in the region of the pelvis minor - like prostatectomies, colorectal cancer surgeries, and gynecological procedures - pose a high risk to pelvic nerves, resulting in urinary or fecal incontinence2. Head and neck surgeries risk damage to cranial nerves, leading to swallowing difficulties, voice changes, or even facial paralysis3.

[0009] Despite advancements in surgical techniques, nerve injuries remain a common source of morbidity, highlighting the need for improved intraoperative nerve visualization and protection. Current methods for nerve identification rely on visual inspection and knowledge of anatomical landmarks, which can be challenging when nerves are obscured by surrounding tissues or pathological change. With minimally invasive approaches in prostate or pelvic surgery, nerves may never be directly visualized and are especially susceptible to blunt or thermal injury when solely relying on anatomical landmarks4. Intraoperative monitoring tools such as electromyographic nerve monitoring only provide feedback after nerve contact or damage has occurred and do not protect sensory nerves5. While efforts to develop novel nerve targeting

[0010] 1

[0011] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0012] DK17352PC agents are underway, no agents have yet received commercial approval, and currently surgeons must rely on visual inspection6,7.

[0013] With current clinical practice, fluorescein via intravenous administration has traditionally been used for ophthalmic angiography but has also been used in peripheral schwannoma resection8,9.

[0014] Nonetheless, there is still a need for improved methods and reagents to reliably visualize sensitive anatomic structures such as nerve structures. This problem is solved by the means and methods of the present invention, with the features of the independent claims. Preferred embodiments, which might be realized in an isolated fashion or in any arbitrary combination are listed in the dependent claims.

[0015] SUMMARY OF THE INVENTION

[0016] In accordance, the present invention relates to a fluorescein dye for use in visualizing an anatomic structure in an operating zone of a subject during a surgical procedure. Preferably, the invention relates to a fluorescein dye for use in visualizing a nerve structure in an operating zone of a subject during a surgical procedure.

[0017] FIGURES

[0018] Fig. 1: Exemplary custom intraoperative fluorescence imaging system. The custom imaging system consists of a movable articulating arm, 2 LEDs for even illumination, and a 12 MP uncooled monochrome camera with a 35 mm lens. The entire system may be covered with a drape to maintain sterility during a surgical procedure.

[0019] Fig. 2a, 2b, and 2c: Visible light and corresponding fluorescence images of exposed CN VII (facial nerve). White solid arrows represent nerves, while the dotted white arrows point to nerve branches not discernable by visible light alone. Star corresponds to lymph node. Scale bar 0.5 cm.

[0020] Fig. 3a-3g: Fluorescence nerve imaging with Zeiss Kinevo Yellow 560 system, a) White light image b) Fluorescence image with Yellow 560 under auto setting c) Fluorescence image with Yellow 560 at shutter setting 1 / 30 d) Contrast optimized Yellow 560 image with Fiji (ImageJ) at shutter setting 1 / 30 e) Close up white light image of the exposed facial nerve f) Contrast optimized Yellow 560 image g) Fluorescence image with custom imaging system. White 2

[0021] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0022] DK17352PC arrows correspond to the main branches of the facial nerve. White dotted arrows refer to small facial nerve branches. White arrow head corresponds to electrocautery char. White star is salivary gland tumor. Linear contrast on images S3d and S3f were adjusted from 0-255 to 0-50 on Fiji.

[0023] Fig. 4a, 4b, and 4c: Colorectal Surgery Robotic da Vinci Imaging. Three surgical fields (A-C) were imaged in white light mode with the Da Vinci endoscope during pelvic surgery (left column) and corresponding fluorescence images with the Da Vinci Firefly fluorescence endoscope (right column). Settings were set at maximum Firefly illumination and lowest white light settings to allow for visualization of nerves of the superior hypogastric plexus otherwise not visible under white light alone. White arrow corresponds to nerves of the superior hypogastric plexus. White star corresponds to the ureter.

[0024] Fig. 5: Pan-body survey of nerves in murine models after administration of sodium fluorescein. Pan-body survey of nerves comparing white light (visible) and fluorescence after fluorescein administration using a custom imaging setup (Example 6). Scale bar 5 mm.

[0025] Fig. 6a and 6b: Porcine facial nerve (arrow) of a pig injected with fluorescein and imaged with a color camera (A) and the custom endoscopic imaging setup B (Example 7).

[0026] Fig, 7a and 7b: Sensitivity comparison of the Da Vinci Firefly endoscope and the custom endoscope using in vitro capillary tubes, a-b) Fluorescence detection of fluorescein dilutions in capillary tubes using (a) the Da Vinci Firefly Xi system and (b) the custom endoscope, (al) Default settings of the Da Vinci generated images dominated by tissue reflections, which prevented the detection of concentrations of 4 pg / ml and lower. (a2) Optimization of the Da Vinci system settings - maximizing Firefly intensity and minimizing white light intensity (optimized settings) - reduced reflections and improved detection of lower concentrations, (b) Fluorescence imaging with the custom endoscope, which contains a band-pass filter optimized for fluorescein imaging, showed further improvements, (bl) When paired with Firefly system illumination, reflections were considerably reduced, and fluorescence contrast was enhanced. (b2) Using custom illumination with additional band-pass filtering to prevent light source detection, reflections were almost entirely eliminated, achieving the highest sensitivity and contrast. Each capillary doublet represents a different concentration of fluorescein diluted in saline; controls included one tube filled with saline and one empty tube. The minimum (min)

[0027] 3

[0028] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0029] DK17352PC and maximum (max) display values, were adjusted on Fiji to enable visualization of lower fluorescein concentrations.

[0030] Fig. 8a and 8b: Comparison of custom endoscope system and Da Vinci Firefly endoscope for embedded fluorescein capillary tube under 1 mm of tissue, a) Fluorescence detection of capillary tube with endoscope using custom illumination source, b) Firefly detection of capillary tube after contrast optimization using Fiji (ImageJ).

[0031] Fig. 9: Comparison of the custom endoscope system of Example 20 and Da Vinci Firefly endoscope in visualization of nerve structures during a rectal resection surgery. Shown are white light illumination (left) vs. fluorescence detection by the Da Vinci system (middle) vs. fluorescence detection by the custom endoscope according to the invention (right).

[0032] Fig. 10a and 10b: Evaluation of fluorescence detection of (a) acute and (b) chronic nerve injury in mice injected with sodium fluorescein, as described in Example 17; points of injury are indicated by thick arrows, nerve structures by thin arrows.

[0033] Fig. Ila and 11b: Fluorescence detection of nerve structures during surgical facial nerve reanimation procedure; two different sections of the operation field are shown (a, b) in both white light and fluorescence detection; solid arrows indicate nerve structures visible under white light, while dotted arrows indicate nerve branches essentially only identifiable through fluorescence imaging.

[0034] Fig. 12a and 12b: Evaluation of fluorescence detection in histopathology and microscopic localization; histological cross-sections of a fresh porcine facial nerve, as specified in Example 19; (a) low- and high-magnification confocal microscopy fluorescence images ; (b) second harmonic generation (SHG) and two-photon fluorescence (TPE), of the high-magnification tissue section in (a). The bright areas in the SHG image correspond to collagen-rich structures; The fluorescein-stained regions (both in (a) and TPE in (b)) are observed in these areas surrounding the nerve fibers, but not in the nerve fibers themselves.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or 4

[0037] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0038] DK17352PC customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, preferably relate to at least one such item, more preferably a plurality thereof; thus, e.g. identifying "a nerve structure" relates to identifying at least one nerve structure, preferably to identifying a multitude of nerve structures.

[0039] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0040] The methods specified herein below, preferably, are in vivo or ex vivo methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, of said steps may be assisted or performed by an electronic and / or (semi-)automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned.

[0041] 5

[0042] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0043] DK17352PC As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component s).

[0044] Unless specifically indicated otherwise herein, the chemical compounds specified may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further chemical molecules, side chains, carrier molecules, retardants, and / or excipients. Also, the chemical compounds referred to herein may be provided as prodrugs, salts, hydrates, solvates, or any other compositions of matter causing the specifically indicated chemical compound to be or become present in the body of a subject, preferably at the indicated dose. The term "prodrug" is understood by the skilled person to relate to a compound not having or having only to a reduced extent the relevant activity of the specifically described chemical compound, but being converted in the body of a subject to a chemical compound having this activity, i.e. preferably to a fluorescein dye as specified. Thus, preferably, a prodrug is an ether or preferably an ester of a chemical compound referred to herein, in particular of a fluorescein dye. In particular, the prodrug of the fluorescein dye may be an ester, e.g. an acetate ester, which does not form the cyclic form as specified herein below. More preferably, the prodrug is a glycosylate e.g. a glucuronide, a phosphate, a sulphate, or a macromolecule-conjugated, e.g. polyethyleneglycol (PEG) conjugated, derivative of the chemical compound, preferably the fluorescein dye. The prodrug can be modified at different positions, e.g. -COOH or -OH of the fluorescent dye.

[0045] 6

[0046] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0047] DK17352PC The term "fluorescein dye" is understood by the skilled person to relate to a group of fluorescent chemical compounds from the group of triarylmethane (or triarylmethine) dyes, specifically xanthene dyes. Preferably, said fluorescein dye has at least one excitation maximum in the range or from 400 nm to 550 nm, preferably of from 420 nm to 530 nm, more preferably of from 425 to 500 nm, even more preferably of from about 450 nm to 495 nm, more preferably of from 480 nm to 495 nm, most preferably of from 451 nm to 487 nm. Also preferably, the fluorescein dye has an emission maximum in the range of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm. Preferably, the fluorescein dye has a structure according to formula (I), preferably formula (la)

[0048]

[0049] wherein each of R1to R5is independently selected from -H, -NCS, halogen, -COOH, C1-C5 alkyl, C1-C5 alkoxy, and -SO3H. More preferably R2to R5are -H and R1is -H. -COOH or NCS, most preferably R1to R5are -H.

[0050] The term "halogen" is understood by the skilled person. Preferably, the halogen is -F, -Cl, or -Br.

[0051] The term “alkyl”, as used herein, refers to a substituted or an unsubstituted, linear or branched, acyclic or cyclic alkyl group, preferably an unsubstituted linear or branched acyclic alkyl group. The alkyl group is a C1-C5 alkyl group, i.e. an alkyl group with one to five carbon atoms.

[0052] The term “alkoxy”, as referred to herein, refers to a substituted or an unsubstituted linear or branched, acyclic or cyclic alkoxy group, preferably an unsubstituted linear or branched acyclic alkoxy group. The alkoxy group is a C1-C5 alkoxy group, i.e. an alkyl group with one to five carbon atoms.

[0053] 7

[0054] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0055] DK17352PC Preferably, the fluorescein dye can exist in two forms, as shown on an exemplary basis for fluorescein in formulas (II) and (III), wherein formula (II) shows the cyclic (lactone) form, and formula (III) shows the open (acid) form.

[0056]

[0057] Formula (II) Formula (III)

[0058] Preferably, the fluorescein dye is fluorescein or a prodrug, salt, or solvate thereof; more preferably, the fluorescein dye is fluorescein (CAS No. 2321-07-5 and / or CAS No. 518-45-6 (open form)) or a salt thereof, preferably sodium fluorescein (CAS No. 518-47-8) or potassium fluorescein (CAS No. 6417-85-2). The fluorescein dye may exist in an open and / or closed form, may be applied as free dye or as a prodrug and be administered in buffer or in a suitable formulation. In a preferred embodiment, sodium fluorescein is used for systemic administration.

[0059] The term "anatomic structure" is understood by the skilled person; preferably, the anatomic structure is a nerve structure, an ureter, and / or a fascia, preferably a fascial plane. Preferably, the anatomic structure is a non-malignant anatomic structure, more preferably is not a tumor. As the skilled person understands, the aforesaid specific anatomic structures were surprisingly found to be stainable with fluorescein dyes by the present invention. Thus, the skilled person understands that these structures can be visualized by the means and methods described herein. However, other anatomic structures may be visualized as well, e.g. by the absence of fluorescein dye in tissue surrounded by tissue stained by the fluorescein dye. Thus, e.g. muscles and blood vessels may be visualized via their surrounding fascia. In a preferred embodiment, the anatomic structure is a tissue rich in connective tissue, preferably is a nerve structure, a fascia, an ureter, and / or an artery, wherein the term "artery" preferably relates to the anatomical structure(s) forming the corresponding blood vessel, but not to its lumen and not to its content, such as blood.

[0060] 8

[0061] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0062] DK17352PC The term "nerve structure", as used herein, relates to each and every structure comprising at least one neuron or a substructure thereof, in particular an axon of a neuron. More preferably, the nerve structure comprises a multitude of neurons or substructures thereof, e.g. at least two, preferably at least three, more preferably at least three, even more preferably at least four, most preferably at least five, neurons or substructures thereof. As the skilled person understands, a nerve structure may comprise further cells or substructures thereof, such as glia cells. Moreover, the nerve structure may comprise further structural elements, such as connective tissue structures such as endoneurium, perineurium and / or epineurium, in a preferred embodiment is an epineurium. Thus, the nerve structure may in particular be a nerve structure such as a nerve fiber, a nerve root, or a ganglion. More preferably, the nerve structure is a nerve fiber. Also preferably, the nerve structure is a nerve structure of the peripheral nervous system, preferably is or comprises an afferent nerve, an efferent nerve, or a mixed nerve. Preferably, the nerve structure is comprised in a body of a subject, i.e. preferably is an in vivo nerve structure. Preferably, the nerve structure is located at least partially within an operating zone during a surgical procedure. Preferably, the nerve structure is a functional nerve structure; i.e. more preferably, is a non-malignant nerve structure.

[0063] Preferably, the nerve structure is at risk of being damaged by said surgical procedure. Preferably, being at risk of being damaged, as referred to herein, relates to a damage rate of at least0.01%, preferably atleastO.1%, more preferably atleast 1%, even more preferably atleast 10%, for a given nerve structure in a given surgical procedure, wherein the term "damage rate" preferably relates to the relative number of subjects suffering from at least symptom caused by loss of conductivity of at least part of said nerve structure caused by said surgical procedure, relative to the number of subjects undergoing said surgical procedure. As the skilled person understands, damage rates may be specific for a type of surgical procedure, for a specific technique applied during said surgical procedure, for a given hospital, and / or even for a specific surgeon. Thus, in case of doubt, the aforesaid damage rates preferably are calculated over a whole region, such as a state, or, more preferably, are calculated over a whole country, or, most preferably, are calculated worldwide, preferably in as far as relevant data are available. The nerve structure preferably is a facial nerve, preferably the nervus facialis, and the surgical procedure is parotidectomy, or the nerve structure is the superior hypogastric plexus and the surgical procedure is colorectal surgery, or the nerve structure comprises nerves of the superior hypogastric plexus and the surgical procedure is partial or total prostatectomy.

[0064] 9

[0065] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0066] DK17352PC In a preferred embodiment, the nerve structure is a damaged nerve structure, i.e. preferably a nerve structure not having normal structure and / or function compared to a corresponding control nerve structure. Causes of nerve structure damage are known in the art and include in particular mechanical causes, such as contusion or cut, and pathological causes, such as insufficient blood supply and / or degenerative nerve diseases. Thus, the anatomic structure in an embodiment is an acutely or chronically damaged nerve structure.

[0067] In concurrence with the above, the term "to avoid damaging" a nerve structure, and grammatical variations thereof, relate to preventing damage to a nerve structure from occurring. As the skilled person understands, avoiding damaging a nerve structure may in particular include avoiding tearing, cutting, cauterizing, stretching or overstretching, of said nerve structure. Preferably, avoiding damaging a nerve structure comprises avoiding cutting through and / or cauterizing said nerve structure. Thus, avoiding damaging a nerve structure preferably comprises avoiding any activity which is known or suspected to prevent, inhibit, or modify nerve conduction through a nerve structure.

[0068] Also preferably, the nerve structure is the or a target of the surgical procedure; thus, the surgical procedure may be or comprise a neurectomy, e.g. a vagotomy, a presacral neurectomy, a vestibular neurectomy, a neurectomy to alleviate nerve entrapment, an intercostal cutaneous nerve neurectomy, or a lateral femoral cutaneous nerve neurectomy. Preferably, the surgical procedure comprises prostate surgery and the anatomic structure is a pelvic nerve, preferably to preserve urinary and sexual function; preferably is gynecological surgery, such as hysterectomy or endometriosis resection and the anatomic structure is a pelvic autonomic nerve, preferably to maintain bowel, bladder, and sexual function; preferably is hip replacement and the anatomic structure is a sciatic or femoral nerve, preferably to prevent nerve injury. Preferably is neurosurgery with involvement of peripheral nerves, preferably to protect or target peripheral nerves, e.g. to ensure optimal outcomes in tumor resection or decompression surgery, preferably is thyroid surgery and the anatomic structure is a recurrent laryngeal nerve, preferably to avoid voice complications; preferably is surgery involving the facial nerve, such as parotidectomy or craniofacial tumor removal, and the anatomic structure is the facial nerve, preferably to avoid facial paralysis, preferably is ear surgery, including procedures like acoustic neuroma removal or cochlear implantation and the anatomic structure is the vestibulocochlear or facial nerve, preferably to minimize complications. The surgical procedure may also aim to identifying sensory nerves that cannot be found by neuromonitoring, such as small or deep 10

[0069] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0070] DK17352PC nerves obscured by fatty, fibrous, or inflamed tissue. Nerve anastomosis surgery may also benefit for acutely and chronically damaged nerves, which requires identification of nerves that may have undergone atrophy.

[0071] The term "ureter" is an anatomic term known to the skilled person, as is the term "fascia".

[0072] The term "operating zone" is understood by the skilled person in the context of the instant description. Preferably, the term includes each and every region on and / or in a body of a subject in which at least one external force is applied during a surgical procedure. Thus, the operating zone may in particular be a region in which surgical instruments, preferably including e.g. a surgeon's fingers and hands, interact, intentionally or non-intentionally, with at least one issue of a subject.

[0073] The term "surgical procedure" is understood by the skilled person. Preferably, the term includes each and every procedure performed on a living body of a subject comprising applying external force to at least one tissue of said subject. Said force may be a mechanical force, such as cutting or tearing a tissue, may be application of thermal, electrical, and / or chemical energy, such as in cauterization, may be application of radiation energy, such as in laser ablation or laser cutting, and the like. The surgical procedure may be of any type deemed appropriate by the skilled person, e.g. an excision, an ablation, a resection, or an incision. Preferably, the surgical procedure is a re-operation, i.e. a further surgical procedure in an operating zone in which a surgical procedure has been performed earlier; such re-operation zones are notorious for sometimes having unpredictable anatomy, e.g. caused by adhesions, scar tissue, and the like. Preferably, the surgical procedure comprises a use of an endoscopic device, preferably is or comprises a laparotomy. Thus, the surgical procedure preferably is a laparoscopic surgical procedure. Also preferably, the surgical procedure is or comprises a salivary gland surgery, preferably salivary gland tumor removal; colorectal surgery, preferably rectum resection; prostate surgery, preferably, partial or complete prostatectomy; gynecological surgery; hip replacement surgery; nerve anastomosis surgery; thyroid surgery, preferably partial or complete thyroidectomy; and / or ear surgery. Also preferably, the surgical procedure is a minimally invasive surgical procedure. Preferably, the surgical procedure is performed at least partially by an electronic and / or (semi-)automated equipment, preferably by a robotic surgical system. More preferably, the surgical procedure is performed by automated equipment, preferably by a robotic surgical system.

[0074] 11

[0075] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0076] DK17352PC

[0077] The term "visualizing" an anatomic structure, preferably a nerve structure, as referred to herein, relates to making the anatomic structure visible in a broad sense, i.e. visible to an eye of a human and / or to a detector as described elsewhere herein. Thus, visualizing may comprise making an anatomic structure detectable for a human eye, but may also comprise, in addition or alternatively, making a nerve structure detectable for an electronic and / or (semi-)automated equipment such as a robotic surgical system, in particular an optical unit thereof. Preferably, visualizing comprises illuminating said operating zone with radiation exciting said fluorescein dye; said illuminating may in particular be required in surgical procedures in which the operating zone is not illuminated with radiation exciting said fluorescein dye anyway. Visualization may be direct to the naked eye or detector; however, visualization may also require further technical equipment, in particular optical equipment, such as a light source and / or at least one filter providing excitation radiation to the operating zone, one or more filters filtering out a specific wavelength or a wavelength range from incident light, one or more filters filtering out a specific wavelength or a wavelength range from light emitted from an operation zone, and / or optical magnification equipment, such as a magnifier or a microscope. Visualization may further require a detector for detecting light emitted from the operating zone; appropriate detectors are known in the art and are described herein below. Also preferably, visualizing comprises identifying at least one anatomic structure within said operating zone. More preferably, in particular in case the surgical procedure is performed at least partially by an electronic and / or (semi-)automated equipment, preferably by a robotic surgical system, visualizing comprises automatically identifying at least one anatomic structure within said operating zone. Methods for automatically identifying anatomic structures are known in the art and include in particular image recognition algorithms, trained automated machine learning models, and the like.

[0078] Preferably, visualizing an anatomic structure comprises administering the fluorescein dye to the subject. The fluorescein dye preferably is administered systemically, e.g. by intravenous administration. Administration may, however also be intraperitoneal or topical, e.g. on the surgical field. Systemic administration preferably comprises administration of the fluorescein dye at a dose of from 0.1 mg / kg body weight to 50 mg / kg body weight, preferably at a dose of from 0.25 mg / kg body weight to 25 mg / kg body weight, still more preferably at a dose of from 0.5 mg / kg body weight to 12.5 mg / kg body weight, even more preferably at a dose of from 2 mg / kg body weight to 10 mg / kg body weight, most preferably at a dose of from 1 mg / kg body 12

[0079] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0080] DK17352PC weight to 5 mg / kg body weight. Topical administration preferably comprises administration of the fluorescein dye at a dose of from 0.1 pg / kg body weight to 50 pg / kg body weight, preferably at a dose of from 0.5 pg / kg body weight to 40 pg / kg body weight, still more preferably at a dose of from 1 pg / kg body weight to 35 pg / kg body weight, even more preferably at a dose of from 5 pg / kg body weight to 30 pg / kg body weight; thus, topic administration preferably comprises administration of a solution of a fluorescein dye having a concentration of from 0.01 mM to 1 mM, preferably of from 0.05 mM to 0.5 mM. Preferably, administration is a one-time administration, although administration may be repeated e.g. once or twice to extend the timeframe of visualization. Preferably, visualizing comprises systemically administering said fluorescein dye at least 30 min before visualizing, preferably at least 60 min before visualizing. Also preferably, visualizing comprises administering said fluorescein dye at most 10 hours before visualization, preferably at most 7.5 hours before visualizing, still more preferably at most 5 hours before visualizing, most preferably at most 3 hours before visualizing.

[0081] Also preferably, visualizing comprises illuminating the operating zone with radiation exciting said fluorescein dye, i.e. preferably causing fluorescence of said fluorescein dye, said radiation preferably having a wavelength in the range of from 400 nm to 500 nm, preferably of from 420 nm to 495 nm, more preferably of from 425 to 495 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 451 nm to 487 nm. Further preferably, visualizing comprises detecting light emitted from the operation zone at a wavelength of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm. Moreover, visualizing preferably comprises avoiding illuminating said operating zone with radiation of a wavelength in the range of from 200 nm to 400 nm, preferably of from 200 nm to 420 nm, more preferably of from 200 nm to 425 nm, even more preferably of from 200 nm to 435 nm; and / or avoiding illuminating said operating zone with radiation of a wavelength in the range of from 500 nm to 550 nm, preferably of from 500 nm to 700 nm.

[0082] The term "subject", as used herein, relates to a vertebrate animal, preferably a mammalian animal, more preferably a livestock, such as a cattle, a pig, a horse, a sheep, or a goat; a pet, such as a rabbit, a guinea pig, or a hare; a companion animal, such as a cat or dog; or a laboratory animal, such as a mouse or a rat. Most preferably, the subject is a human.

[0083] Advantageously, it was found in the work underlying the present invention that fluorescein dyes can be used effectively to make e.g. nerve structures in a body of a subject detectable during 13

[0084] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0085] DK17352PC surgical procedures and that this visualization is possible even when the nerve structure is obscured e.g. by fat tissue. Moreover, it was found that the sensitivity of nerve structure detection can be significantly improved by modifying existing optical equipment.

[0086] The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

[0087] The present invention further relates to a method for visualizing an anatomic structure in an operating zone of a subject during a surgical procedure comprising

[0088] (i) contacting said nerve structure with a fluorescein dye;

[0089] (ii) illuminating said operating zone with light exciting said fluorescein dye; and

[0090] (iii) thereby visualizing said nerve structure.

[0091] In a preferred embodiment, the present invention also relates to a method of histopathologically visualizing an anatomic structure rich in connective tissue, comprising

[0092] (I) contacting said anatomic structure with a fluorescein dye,

[0093] (II) illuminating said anatomic structure with light exciting said fluorescein dye; and

[0094] (III) thereby visualizing said anatomic structure.

[0095] In a preferred embodiment, in the histopathological method, the anatomic structure which is visualized is a tissue rich in connective tissue, preferably is a nerve structure, a fascia, an ureter, and / or an artery. In a preferred embodiment, the histopathological method is performed in vitro or in vivo, more preferably of performed in vitro on a sample of a subject.

[0096] The term “contacting”, as used in the context of the methods of the present description, is understood by the skilled person. The term preferably relates to bringing a compound, in particular a fluorescein dye, in physical contact with a subject or a sample, thereby allowing the compound to interact with constituents of the subject or the sample. Preferably, contacting is administering the fluorescein dye to a subject, as specified herein above.

[0097] The present invention also relates to a method for treating a subject by a surgical procedure comprising

[0098] 14

[0099] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0100] DK17352PC (A) visualizing an anatomic structure in an operating zone of said subject during said surgical procedure according to the method specified herein above; and

[0101] (B) thereby treating said subject by said surgical procedure.

[0102] The term “treating” refers to an amelioration of a disease or disorder referred to herein or the symptoms accompanied therewith, preferably to a significant extent. Said treating as used herein also includes an entire restoration of the health with respect to the diseases or disorders referred to herein. It is to be understood that treating as used in accordance with the present invention may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test etc. Preferred confidence intervals are at least 90 %, at least 95 %, at least 97 %, at least 98 % or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 10 %, at least 20 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, or at least 90 % of the subjects of a given cohort or population. Also, the skilled person will understand that e.g. avoiding damaging an anatomic structure may not be possible in all subjects to be treated. However, with the methods and devices described herein, preferably a statistically significant portion of anatomic structure damaging events shall be successfully avoided.

[0103] The present invention also relates to a method for staining an anatomic structure, comprising (I) contacting said anatomic structure with a fluorescein dye, and

[0104] (II) thereby staining said anatomic structure.

[0105] The method for staining an anatomic structure may be an in vitro or ex vivo method, e.g. applied to an isolated sample known or suspected to comprise an anatomic structure of interest; the method for staining an anatomic structure may, however, also be an in vivo method, e.g. as part of the method for visualizing an anatomic structure in an operating zone and / or the method for treating a subject by a surgical procedure described herein above. Moreover, the method for staining an anatomic structure may also be comprised in a method of cosmetic treatment of a subject, wherein the term "cosmetic treatment" preferably relates to a method of treating a subject's body without health benefit; thus, the cosmetic treatment may e.g. be administration 15

[0106] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0107] DK17352PC of a tattoo. As indicated herein above, the method may comprise additional steps, e.g. of removing excess fluorescein dye.

[0108] The present invention also relates to a surgical detection device comprising an illumination unit adapted for at least partially illuminating an operating zone of a subject with radiation exciting a fluorescein dye.

[0109] The term “surgical detection device”, as used herein, relates to a system of means comprising at least the means described, operatively linked to each other as to allow the visualization and, optionally, detection. How to link the means of the device in an operating manner will depend on the type of means included into the device. Preferably, the means are comprised by a single device. However, it is also contemplated that the means of device may appear as separate units and are, preferably, packaged together as a kit. The person skilled in the art will realize how to link the means without further ado. Preferred devices are those which can be applied without the particular knowledge of a specialized technician. Preferably, the device is adapted to include an additional feature as described herein.

[0110] The surgical detection device includes an illumination unit adapted for at least partially illuminating an operating zone of a subject with radiation exciting a fluorescein dye. As specified in more detailed herein elsewhere herein; in said illuminating it is preferably avoided to illuminate said operating zone with radiation of a wavelength in the range of from 200 nm to 400 nm, preferably of from 200 nm to 420 nm, more preferably of from 200 nm to 425 nm, even more preferably of from 200 nm to 435 nm; and / or avoiding illuminating said operating zone with radiation of a wavelength in the range of from 500 nm to 550 nm, preferably of from 500 nm to 700 nm.

[0111] Thus, the illumination unit comprises (i) a radiation source emitting radiation having a wavelength in the range of from 400 nm to 550 nm, preferably of from 420 nm to 530 nm, more preferably of from 425 to 500 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 451 nm to 487 nm. As the skilled person understands, the radiation source does not have to, but may, be preferred to emit radiation predominantly or exclusively in one of the aforesaid wavelength ranges, wherein "predominantly" emitting in a given wavelength range relates to emitting at least 75% of the total energy emitted in said wavelength range, and wherein "exclusively" emitting in a given wavelength range relates to emitting at least 95%,

[0112] 16

[0113] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0114] DK17352PC preferably at least 99%, of the total energy emitted in said wavelength range. Thus, the radiation source may be a standard white light source, e.g. of an endoscopic device or of a robotic surgical device; the radiation source may, however, also be e.g. a 470 nm or 488 nm LED light source. In a preferred embodiment, the radiation source comprises a laser, preferably a laser emitting radiation in the aforesaid range, more preferably at 488 nm.

[0115] The illumination unit optionally further comprises (ii) a short-pass filter with a cutoff wavelength in the range 480 nm to 495 nm, (iii) a long-pass filter with a cutoff wavelength in the range of from 400 nm to 450 nm; and / or (iv) a band-pass filter with a central wavelength in the range of from 450 nm to 490 nm and with a bandwidth in the range of from 50 nm to 30 nm. Thus, the band-pass filter (iv) may e.g. be a 469-35 or a 469-50 filter. As the skilled person understands, the filters (ii) to (iv), if present, are arranged to intervene between the radiation source and the operating zone.

[0116] As the skilled person will understand in view of the description herein, a surgical detection device comprising only an illumination unit may be used to visualize anatomic structures e.g. to a human operator. In a preferred embodiment, it is envisaged that the surgical detection device comprises, alternatively or in addition, at least one detection filter as described herein below for the detection unit, preferably a filter filtering out excitation radiation. Thus, in a preferred embodiment, the surgical detection device is configured for detection of a fluorescein dye by an operator, e.g. a medical practitioner, preferably is a pair of glasses or a surgical loupe equipped with light sources and / or filters as specified herein elsewhere and as shown in e.g. Zhang et al. (2024), JNeurosurg Sci 67(3):374.

[0117] Preferably, the surgical detection device further comprises a detector unit, preferably adapted for detecting radiation emitted from a fluorescein dye in an operating zone. Thus, the detector unit may in particular comprise a detector of fluorescence of a fluorescein dye. Thus, the detector unit preferably comprises (aa) a detector adapted for detecting radiation with a wavelength in the range of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm. Exemplary detectors are configured for converting electromagnetic energy into an electrical signal, which includes both single and multi-element or array optical detectors. As referred to herein, a detector is preferably capable of monitoring an optical electro-magnetic signal and providing an electrical outlet signal or response signal relative to a baseline signal indicative of fluorescent radiation emitted by a fluorescein dye. Such devices may include, for example,

[0118] 17

[0119] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0120] DK17352PC photodiodes, including avalanche photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS detectors, including CMOS array detectors, photomultipliers, and photomultiplier arrays. Thus, the detector may in particular be a two-dimensional or three-dimensional resolving detector, preferably adapted to provide information on the location of an anatomic structure in an operation zone. Also, the detector, such as a photodiode or photomultiplier, may contain additional signal conditioning or processing electronics. For example, an optical detector may include at least one pre-amplifier, electronic filter, or integrated circuit. Moreover, the detector and / or the detector unit is preferably operably linked to at least one processor, e.g. a processor of the surgical detection device. The detector and / or the detector unit may, however, also be operably linked to a microprocessor of at least one further device, such as a robotic surgical device, e.g. in a system as referred to herein, or to a microprocessor of e.g. a computer.

[0121] The detector unit of the surgical detection device may optionally further comprise (bb) a long-pass filter with a cutoff wavelength in the range of from 490 nm to 500 nm, preferably of from 495 nm to 500 nm; (cc) a short pass filter with a cutoff wavelength in the range of from 540 to 600 nm, preferably 550 nm to 580 nm, more preferably 550 nm; and / or (dd) a band-pass filter with a central wavelength in the range of from 520 nm to 550 nm and with a bandwidth of from 50 nm to 30 nm. Thus, the band pass filter (dd) may e.g. be a 520-36 or a 525-50 filter. Preferably, said filters (bb), (cc), and / or (dd) if present, are arranged to intervene between the operating zone and the detector unit.

[0122] In a preferred embodiment, the surgical detection device, preferably the detector unit thereof, comprises a band stop filter (notch filter) filtering out excitation radiation, preferably radiation in the range of 420 nm to 500 nm, preferably of from 440 nm to 500 nm, more preferably of from 450 to 500 nm, even more preferably of from about 460 nm to 500 nm, most preferably of from 481 nm to 495 nm. Thus, the notch filter may e.g. be a filter NF488-14 (Semrock).

[0123] In a preferred embodiment, the illumination unit described herein above comprises a white light source, wherein the term "white light" is used in a broad sense encompassing any light conveying to an observer the impression of an essentially white light. Thus, the white light in a preferred embodiment comprises radiation in the ranges of red light, green light, and blue light, preferably further comprises radiation in the far-red and / or near infrared range(s). Preferably, the white light comprises radiation of the wavelengths of from 410 nm to 800 nm, such as e.g.

[0124] 18

[0125] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0126] DK17352PC the Thorlabs MNWHL4 light source. In a further preferred embodiment, the surgical detection device comprises an illumination unit configured to provide pulsed illumination of the operating zone and / or is configured for pulsed detection, wherein said pulsed illumination and pulsed detection preferably are synchronized. Said illumination unit in a preferred embodiment is configured to at least partially illuminate the operating zone with white light, preferably as sole radiation source, as an additional radiation source, or, more preferably, as an alternative radiation source. Thus, the surgical detection device preferably comprises a detector unit, wherein the aforesaid pulsed illumination is synchronized with detecting said radiation, in a preferred embodiment wherein said pulsed illumination and said detecting radiation are performed at a rate of video capture, preferably of from 10Hz to 100Hz, preferably of from 25 Hz to 84 Hz. Preferably, illumination with radiation having a wavelength in the range of from 400 nm to 500 nm and illumination with white light are performed essentially alternatively, more preferably are performed alternatively, i.e. preferably one picture is acquired by the detector unit per one illumination pulse. As referred to herein, the term "essentially alternatively" relates to an illumination sequence in which illumination with a white light (W) and the radiation exciting a fluorescein dye (F) alternate in a repeating pattern, preferably [WF]n, wherein n is an integer of from 1 to infinite; the skilled person knows that the value of n will essentially be determined by the frequency of alternations and duration of detection. However, also other patterns may be envisaged, such as [WFF]n, [WWF]n, [WWFF]n, and the like. Also, one or more additional detections may be performed without the aforesaid light sources in order to detect a background signal; said background signal, captured e.g. as one or more background frame, preferably is subtracted from the detections made under illumination.

[0127] In an exemplary embodiment, the surgical detection device comprises a CMOS sensor and optionally a 35-mm camera lens; also preferably, the emission pathway comprises three main filters: a long-pass (LP) 500 nm filter, a color LP filter 495 nm, and a short-pass (SP) 550 nm filter; also preferably, illumination is provided by at least one, preferably two, 470 nm LEDs, optionally with a maximum power density of 12 mW / cm2across the field of view. Also preferably, each LED is equipped with a lens positioned slightly out of focus or using engineered diffusers to create even illumination. Also preferably, the surgical detection device in its excitation pathway further comprises a clean-up band-pass (BP) filter 469-35, represented as central wavelength - full width at half maximum. Also preferably, the surgical detection device is adapted to save images as 12 bit (0 to 4095).

[0128] 19

[0129] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0130] DK17352PC In a further exemplary embodiment, the surgical detection device comprises an endoscope with a 10-mm diameter and 30° head angle, connected to a CMOS camera and a Zoom Adapter. Preferably, the surgical detection device comprises an illumination unit comprising a 470-nm LED, a 469 nm band pass filter with a 35-nm band width, and a liquid light guide to the endoscope; preferably, the surgical detection device is adapted to provide a maximum power density between 2.5 and 8 mW / cm2. Also preferably, the surgical detection device further comprises a 520 nm band pass filter with a 36-nm band width attached to the camera adapter.

[0131] Preferably, the surgical detection device comprises at least one processor, adapted for performing a method described herein.

[0132] The term “processor”, as used herein, relates to an arbitrary logic circuitry configured for performing basic operations of a device, computer, or system, and / or, generally, to a device which is configured for performing calculations or logic operations. In particular, the processing unit may be configured for processing basic instructions that drive the device, computer, or system. As an example, the processor may comprise at least one arithmetic logic unit (ALU), at least one floating-point unit (FPU), such as a math co-processor or a numeric coprocessor, a plurality of registers, specifically registers configured for supplying operands to the ALU and storing results of operations, and a memory, such as an LI and L2 cache memory. In particular, the processor may be a multi-core processor. Specifically, the processing unit may be or may comprise a central processing unit (CPU). Additionally or alternatively, the processor may be or may comprise a microprocessor, thus specifically the processing unit’s elements may be contained in one single integrated circuitry (IC) chip. Additionally or alternatively, the processing unit may be or may comprise one or more application-specific integrated circuits (ASICs) and / or one or more field-programmable gate arrays (FPGAs) or the like. The processing unit specifically may be configured, such as by software programming, for performing one or more evaluation operations, preferably as described elsewhere herein.

[0133] The present invention also relates to a surgical system comprising (a) a surgical detection device as described herein above and (B) a robotic surgical device.

[0134] Robotic surgical devices are known in the art and can be provided by the skilled person without further ado. Preferred robotic surgical devices are those that are commercially available. As the skilled person will understand in view of the description herein, robotic surgical devices may 20

[0135] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0136] DK17352PC already comprise or may be operably linked to detection devices comprising an illumination unit and, optionally, a detector unit. However, these typically require at least one modification to provide a surgical system as described herein. In particular, the elements of the illumination unit and, optionally, the detector unit of the surgical detection device will have to be provided. To that end, the elements in the existing robotic surgical device may be modified as described herein; or the surgical detection device of the present description may be added or may be used to replace any existing surgical detection device. Preferably, the elements of the surgical detection device described herein are added to the robotic surgical device, wherein "adding" structural and / or functional elements may relate to physically adding said elements to a robotic surgical device, or may relate to providing an operable link between the robotic surgical device and the surgical detection device, preferably in a manner allowing the surgical detection device or data provided therefrom to be used to at least partially control the robotic surgical device. Thus, the surgical detection device may e.g. provide data to the robotic surgical device about anatomic structures to avoid and / or to aim at.

[0137] To that end, the surgical detection device and / or the surgical system may comprise an evaluation unit, wherein the term "evaluation unit" preferably includes each and every device adapted for analyzing data provided by a detector unit as described herein to visualize and, preferably, detect at least one anatomic structure. The evaluation unit preferably comprises a memory unit comprising tangibly embedded an algorithm for visualizing and, preferably, detecting at least one anatomic structure. More preferably, the tangibly embedded algorithm implements the visualization and, preferably, detection of at least one anatomic structure according to a method of the present invention.

[0138] The present invention also relates to a kit for visualizing an anatomic structure in an operating zone during a surgical procedure, the kit comprising (AA) a fluorescein dye and (BB) a surgical detection device as described herein; and / or a surgical system as described herein; and / or a manual comprising instructions for performing a method as described herein.

[0139] The term “kit”, as used herein, refers to a collection of the aforementioned compounds and / or means which may or may not be packaged together. The components of the kit may be comprised by separate containers (i.e. as a kit of separate parts) or provided in a single container. The housing of the kit preferably allows translocation of the compounds of the kit, in particular common translocation; thus, the housing may in particular be a transportable container 21

[0140] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0141] DK17352PC comprising all specified components. Moreover, it is to be understood that the kit of the present invention may be used for practicing the methods referred to herein above. It is, preferably, envisaged that all components are provided in a ready -to-use manner for practicing the methods referred to above. Further, the kit preferably contains instructions for carrying out said methods. The instructions can be provided by a user's manual on paper or in electronic form. For example, the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit. Preferably, the kit comprises further compounds, such as a means of administration of the fluorescein dye, a solvent for dissolving the fluorescein dye, and the like. Preferably, the kit is adapted for use in a method of the present invention, more preferably is adapted to comprise all reagents required to perform said method or methods.

[0142] The present invention also relates to a use of a fluorescein dye for the manufacture of a diagnostic for visualizing an anatomic structure in an operating zone during a surgical procedure; and to a use of a fluorescein dye for visualizing an anatomic structure in an operating zone during a surgical procedure.

[0143] In view of the above, the following embodiments are particularly envisaged:

[0144] Embodiment 1 : A fluorescein dye for use in visualizing an anatomic structure in an operating zone of a subject during a surgical procedure.

[0145] Embodiment 2: The fluorescein dye for use of embodiment 1, wherein said visualizing further comprises illuminating said operating zone with radiation exciting said fluorescein dye. Embodiment 3: A method for visualizing an anatomic structure in an operating zone of a subject during a surgical procedure comprising

[0146] (i) contacting said anatomic structure with a fluorescein dye;

[0147] (ii) illuminating said operating zone with light exciting said fluorescein; and

[0148] (iii) thereby visualizing said anatomic structure.

[0149] Embodiment 4: The subject matter of any one of embodiments 1 to 3, wherein said visualizing comprises administering said fluorescein dye to said subject at a dose of from 0.1 mg / kg body weight to 50 mg / kg body weight, preferably at a dose of from 0.25 mg / kg body weight to 25 mg / kg body weight, still more preferably at a dose of from 0.5 mg / kg body weight to 12.5 mg / kg body weight, even more preferably at a dose of from 2 mg / kg body weight to 10 mg / kg body weight, most preferably at a dose of from 1 mg / kg body weight to 5 mg / kg body weight, wherein said fluorescein dye preferably is administered systemically.

[0150] 22

[0151] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0152] DK17352PC Embodiment s: The subject matter of any one of embodiments 1 to 4, wherein said visualizing comprises administering said fluorescein dye at least 30 min before visualizing, preferably at least 60 min before visualizing.

[0153] Embodiment 6: The subject matter of any one of embodiments 1 to 5, wherein said visualizing comprises administering said fluorescein dye at most 10 hours before visualization, preferably at most 7.5 hours before visualizing, still more preferably at most 5 hours before visualizing, most preferably at most 3 hours before visualizing.

[0154] Embodiment 7: The subject matter of any one of embodiments 1 to 6, wherein said surgical procedure comprises a use of an endoscopic device, preferably is or comprises a laparotomy.

[0155] Embodiment s: The subject matter of any one of embodiments 1 to 7, wherein said visualizing comprises illuminating said operating zone with radiation having a wavelength in the range of from 400 nm to 500 nm, preferably of from 420 nm to 495 nm, more preferably of from 425 to 495 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 451 nm to 487 nm.

[0156] Embodiment 9: The subject matter of any one of embodiments 1 to 8, wherein said visualizing comprises detecting light emitted from the operation zone at a wavelength of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm.

[0157] Embodiment 10: The subject matter of any one of embodiments 1 to 9, wherein said visualizing comprises avoiding illuminating said operating zone with radiation of a wavelength in the range of from 200 nm to 400 nm, preferably of from 200 nm to 420 nm, more preferably of from 200 nm to 425 nm, even more preferably of from 200 nm to 435 nm; and / or avoiding illuminating said operating zone with radiation of a wavelength in the range of from 500 nm to 550 nm, preferably of from 500 nm to 700 nm.

[0158] Embodiment 11: The subject matter of any one of embodiments 1 to 10, wherein said anatomic structure is a nerve structure, an ureter, and / or a fascia, preferably a fascial plane; in a preferred embodiment wherein said anatomic structure is a tissue rich in connective tissue, preferably is a nerve structure, a fascia, an ureter, and / or an artery.

[0159] Embodiment 12: The subject matter of any one of embodiments 1 to 11, wherein said visualizing comprises identifying at least one anatomic structure within said operating zone. Embodiment 13: The subject matter of any one of embodiments 1 to 12, wherein said anatomic structure is a nerve structure and wherein said surgical procedure is or comprises a salivary gland surgery, colorectal surgery, prostate surgery, gynecological surgery, mastectomy, hip replacement surgery, nerve anastomosis surgery, thyroid surgery, and / or ear 23

[0160] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0161] DK17352PC surgery; in a preferred embodiment wherein said anatomic structure is an acutely or chronically damaged nerve structure.

[0162] Embodiment 14: The subject matter of any one of embodiments 1 to 12, wherein said anatomic structure is an ureter and wherein said surgical procedure is or comprises colorectal surgery, prostate surgery, gynecological surgery.

[0163] Embodiment 15: The subject matter of any one of embodiments 1 to 12, wherein said anatomic structure is a fascia.

[0164] Embodiment 16: The subject matter of any one of embodiments 1 to 15, wherein said surgical procedure is a laparoscopic surgical procedure.

[0165] Embodiment 17: The subject matter of any one of embodiments 1 to 16, wherein said surgical procedure is a minimally invasive surgical procedure.

[0166] Embodiment 18: The subject matter of any one of embodiments 1 to 17, wherein said method comprises automatically identifying at least one anatomic structure within said operating zone.

[0167] Embodiment 19: The subject matter of any one of embodiments 1 to 18, wherein said surgical procedure is performed at least partially by an electronic and / or semi-automated or automated equipment, preferably by a robotic surgical system.

[0168] Embodiment 20: The subject matter of any one of embodiments 1 to 19, wherein damaging said anatomic structure visualized is avoided.

[0169] Embodiment 21: The subject matter of any one of embodiments 1 to 20, wherein said anatomic structure is a nerve structure and wherein said nerve structure is a target of a neurectomy, preferably wherein said surgical procedure is vagotomy, presacral neurectomy, vestibular neurectomy, neurectomy to alleviate nerve entrapment, intercostal cutaneous nerve neurectomy, or lateral femoral cutaneous nerve neurectomy.

[0170] Embodiment 22: A method for staining an anatomic structure, comprising

[0171] (I) contacting said anatomic structure with a fluorescein dye, and

[0172] (II) thereby staining said anatomic structure.

[0173] Embodiment 23: The method of embodiment 22, wherein said method further comprises a step of removing excess fluorescein dye.

[0174] Embodiment 24: The method of embodiment 22 or 23, wherein said method is an ex vivo and / or in vitro method or is a cosmetic method.

[0175] Embodiment 25: A method for treating a subject by a surgical procedure comprising (A) visualizing an anatomic structure in an operating zone of said subject during said surgical procedure according to the method according to any one of embodiments 3 to 25; and 24

[0176] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0177] DK17352PC (B) thereby treating said subject by said surgical procedure.

[0178] Embodiment 26: The method of embodiment 25, further comprising detecting at least one nerve structure in an operating zone and avoiding damaging the nerve structure detected; and / or further comprising detecting at least one ureter in an operating zone and avoiding damaging the ureter detected.

[0179] Embodiment 27: The subject matter of any one of embodiments 1 to 26, wherein said fluorescein dye has a structure according to formula (I), preferably formula (la)

[0180]

[0181] wherein each of R1to R5is independently selected from -H, -COOH, -NCS, halogen, C1-C5 alkyl, C1-C5 alkoxy, and -SO3H, preferably is fluorescein (CAS No. 2321-07-5 and / or CAS No.

[0182] 518-45-6) or a salt thereof, preferably sodium fluorescein (CAS No. 518-47-8) or potassium fluorescein (CAS NO. 6417-85-2).

[0183] Embodiment 28: The subject matter of any one of embodiments 1 to 27, wherein said anatomic structure is a nerve structure of the peripheral nervous system, preferably an afferent, efferent, or mixed nerve.

[0184] Embodiment 29: The subject matter of any one of embodiments 1 to 28, wherein said anatomic structure is a nerve fiber, a nerve root, a ganglion.

[0185] Embodiment 30: A surgical detection device comprising an illumination unit adapted for at least partially illuminating an operating zone of a subject with radiation exciting a fluorescein dye.

[0186] Embodiment 31 : The surgical detection device of embodiment 30, wherein said illumination unit comprises

[0187] (i) a radiation source emitting radiation having a wavelength in the range of from 400 nm to 550 nm, preferably of from 420 nm to 530 nm, more preferably of from 425 to 500 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 451 nm to 487 nm; and, optionally,

[0188] (ii) a short-pass filter with a cutoff wavelength in the range 480 nm to 495 nm,

[0189] 25

[0190] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0191] DK17352PC (iii) a long-pass filter with a cutoff wavelength in the range of from 400 nm to 450 nm; and / or (iv) a band-pass filter with a central wavelength in the range of from 450 nm to 490 nm and with a bandwidth in the range of from 50 nm to 10 nm;

[0192] wherein said filters (ii) to (iv), if present, are arranged to intervene between the radiation source and the operating zone.

[0193] Embodiment 32: The surgical detection device of embodiment 30 or 31, wherein said illumination unit comprises at least said radiation source and, at least one, preferably two, of said filters (ii) to (iv).

[0194] Embodiment 33: The surgical detection device of any one of embodiments 30 to 32, wherein said surgical device further comprises a detector unit comprising

[0195] (aa) a detector adapted for detecting radiation with a wavelength in the range of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm; and, optionally,

[0196] (bb) a long-pass filter with a cutoff wavelength in the range of from 490 nm to 500 nm, preferably of from 495 nm to 500 nm; (cc) a short pass filter with a cutoff wavelength in the range of from 540 to 600 nm, preferably 550 nm to 580 nm, more preferably 550 nm; and / or (dd) a band-pass filter with a central wavelength in the range of from 520 nm to 550 nm and with a bandwidth of from 50 nm to 10 nm;

[0197] wherein said filters (bb), (cc) and (dd), if present, are arranged to intervene between the operating zone and the detector unit.

[0198] Embodiment 34: The surgical detection device of any one of embodiments 30 to 33, wherein said surgical detection device further comprises at least one processor adapted for performing the method according to any one of the preceding embodiments relating to a method.

[0199] Embodiment 35: The surgical detection device of any one of embodiments 30 to 34, wherein said surgical detection device is adapted for automatically detecting at least one anatomic structure in an operating zone.

[0200] Embodiment 36: The surgical detection device of any one of embodiments 30 to 35, comprising a band stop filter filtering out radiation in the range of 420 nm to 500 nm, preferably of from 440 nm to 500 nm, more preferably of from 450 to 500 nm, even more preferably of from about 460 nm to 500 nm, most preferably of from 481 nm to 495 nm

[0201] Embodiment 37: The surgical detection device of any one of embodiments 30 to 36, wherein said illumination unit is configured to provide pulsed illumination of the operating zone.

[0202] 26

[0203] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0204] DK17352PC Embodiment 38: The surgical detection device of any one of embodiments 30 to 37, comprising an illumination unit at least partially illuminating the operating zone with white light.

[0205] Embodiment 39: The surgical detection device of embodiment 37 or 38 comprising a detector unit as specified in embodiment 33, wherein said pulsed illumination is synchronized with said detecting radiation, in a preferred embodiment wherein said pulsed illumination and said detecting radiation are performed at a rate of video capture, preferably of from 10Hz to 100Hz.

[0206] Embodiment 40: The surgical detection device of embodiment 39, wherein illumination with radiation having a wavelength in the range of from 400 nm to 500 nm and illumination with white light are performed essentially alternatively.

[0207] Embodiment 41: The surgical detection device of any one of embodiments 37 to 40, wherein one image is captured for each illumination pulse, in a preferred embodiment by the detector unit.

[0208] Embodiment 42: A surgical system comprising (a) a surgical detection device according to any one of embodiments 30 to 35 and (B) a robotic surgical device.

[0209] Embodiment 43 : The surgical system of embodiment 36, wherein the surgical detection device is adapted for automatically detecting at least one anatomic structure in an operating zone, and wherein said robotic surgical device is adapted to avoid damaging the anatomic structure detected by said surgical detection device.

[0210] Embodiment 44: A kit for visualizing an anatomic structure in an operating zone during a surgical procedure, the kit comprising (AA) a fluorescein dye and (BB) a surgical detection device according to any one of embodiments 30 to 35 and / or a surgical system according to embodiment 36 or 37; and / or a manual comprising instructions for performing a method according to any of the preceding embodiments reflating to a method.

[0211] Embodiment 45: Use of a fluorescein dye for the manufacture of a diagnostic for visualizing an anatomic structure in an operating zone during a surgical procedure.

[0212] Embodiment 46: Use of a fluorescein dye for visualizing an anatomic structure in an operating zone during a surgical procedure.

[0213] Embodiment 47: The subject matter of any of the preceding embodiments, wherein damage to said anatomic structure is avoided by said visualizing.

[0214] Embodiment 48: The subject matter of any of the preceding embodiments, wherein said subject is a mammal, preferably a human.

[0215] 27

[0216] 4917-8732-8394.1Attorney Ref.: 097147-0181

[0217] DK17352PC Embodiment 49: The subject matter of any of the preceding embodiments referring to a method, wherein said method is performed using a surgical detection device according to any one of embodiments 30 to 35 and / or a surgical system according to embodiment 36 or 37. Embodiment 50: The method of any one of embodiments 22 to 24, wherein said anatomic structure is a tissue rich in connective tissue, preferably is a nerve structure, a fascia, and ureter, and / or an artery.

[0218] Embodiment 51: A method of histopathologically visualizing an anatomic structure rich in connective tissue, comprising

[0219] (I) contacting said anatomic structure with a fluorescein dye,

[0220] (II) illuminating said anatomic structure with light exciting said fluorescein; and

[0221] (III) thereby visualizing said anatomic structure..

[0222] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0223] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0224] Example 1: Custom Clinical Fluorescence Imaging Set Up

[0225] The imaging setup (Figure 1) utilized a Teledyne FLIR Blackfly S USB3 12 MP camera (BFS-U3-120S6M-C) controlled via MicroManager. The camera operated without cooling, gain, or gamma adjustment (gamma set to 1) with binning set at 2. A 35-mm camera lens (f / 1.4, Thorlabs, catalog no. MVL35M1) was used. The emission pathway was designed for optimal transmission in the range between 500 and 550 nm, achieved using three main filters: a long-pass (LP) 500 nm filter (Thorlabs, catalog no. FELH0500), color LP filter 495 nm (Thorlabs, catalog no. FGL495M), and a short-pass (SP) 550 nm filter (Thorlabs, catalog no. FESH0550). Illumination was delivered by two 470 nm LEDs (M470L5, Thorlabs) with a maximum power density of 12 mW / cm2across the field of view. Each LED was equipped with an ACL2520U lens, positioned slightly out of focus to create even illumination. A clean-up band-pass (BP) filter 469-35, represented as central wavelength - full width at half maximum (Thorlabs, catalog no. MF469-35) further refined the excitation wavelength range. Images were saved as 12 bit (0 to 4095). Visible images were taken with an Allied Vision Alvium 12 MP color camera (1800

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[0228] DK17352PC U-1240c) with a 25 mm camera lens (fl .4, Thorlabs, catalog no. MVL25M23) or Nikon DSLR 3300 camera with a 35 mm lens.

[0229] Example 2: Head and Neck Nerve Imaging Clinical Trial

[0230] The clinical trial was approved by the Stanford Institutional Review Board (IRB 71857), Stanford Cancer Institute (SCI) Scientific Research Committee, and the study was registered on ClinicalTrials.gov (ClinicalTrials.gov identifier: NCT06054178). Eligible patients undergoing head and neck surgery older than 18 years provided written informed consent. Patients with history of fluorescein allergy or history of kidney disease were not eligible to participate in the study. No adverse effects related to dye administration were observed for enrolled patients in the study.

[0231] Upon induction of anesthesia, patients were administered an intravenous dose of 1 mg / kg sodium fluorescein (Fluorescite Injection 10%). All medications were obtained from the Stanford Investigational Drug Service (IDS) pharmacy. No long-term paralytics were administered to allow for nerve monitoring, and sedation levels were maintained with total intravenous anesthesia (TIVA). Facial nerve function was monitored intraoperatively via electromyography (EMG) with the NIM nerve vital monitoring system (Medtronic). Following dissection and identification of the main trunk of the facial nerve, nerve imaging was performed with our custom imaging system at an exposure time of 50 to 100 ms (Figure 2). Nerve branches identified on the custom imaging system that were not visible with white light were further verified with EMG stimulation at 0.5 mA. To prevent any light contamination, all computer screens and overhead lights were turned off during fluorescence imaging.

[0232] As a benchmark, the Yellow 560 fluorescence system on the Zeiss Kinevo was also compared to our custom system intraoperatively. Images were acquired at both auto and adjusted manual settings. The best fluorescence images on the Yellow 560 system were observed with the manual shutter setting at 1 / 30 (Figure 3).

[0233] Example 3: Colorectal Surgery Robotic da Vinci Imaging

[0234] Informed written consent with explicit consent for publication of data was obtained from a single patient undergoing colorectal robotic surgery with the Da Vinci Xi at the University Hospital Carl Gustav Cams of the Technical University Dresden. Following induction of anesthesia, the patient was administered an intravenous dose of 5 mg / kg sodium fluorescein 29

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[0236] DK17352PC (Fluorescite Injection 10%). Fluorescence imaging was performed with the Da Vinci Xi Firefly Fluorescence system on Standard mode with settings on lowest white light and highest Firefly fluorescence intensities (Figure 4). No adverse effects from fluorescein administration were observed.

[0237] Example 4: Animal Imaging

[0238] Murine protocols were approved by the Stanford Administrative Panel on Laboratory Animal Care. Further murine protocols were approved by the government of Upper Bavaria, Germany. Porcine experiments were approved by the government of Saxony, Germany. All procedures complied with ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines.

[0239] Example 5: Murine Fluorescein Studies

[0240] 6 to 8 week old balb / c mice (Jackson Laboratory) were anesthetized with 2% v / v isoflurane and intravenously administered 15 nmol / gram sodium fluorescein (Sigma-Aldrich) via retro-orbital injection. 5 hours following administration of sodium fluorescein (n = 5), mice were euthanized with carbon dioxide in a compressed chamber provided by the Stanford animal research facility. Following this, pan-body surveys of nerves were performed with both white light and fluorescence imaging (Figure 5). All imaging for the murine experiments were performed using custom set up (Imaging Setup A). All images were acquired at 100 ms.

[0241] Example 6: Imaging Setup A (Murine Studies)

[0242] For murine studies, the same optical set up as the custom clinical intraoperative imaging device was used. All visible light images were taken with the Allied Vision Alvium 12 MP color camera (1800 U-1240c) with a 25 mm camera lens (fl.4, Thorlabs, catalog no. MVL25M23) on Vimba Viewer X (Allied Vision). For fluorescence imaging, the illumination was delivered with a maximum power density of 25 mW / cm2across the field of view.

[0243] Example 7: Imaging Setup B (Custom Endoscope)

[0244] This custom fluorescein endoscope consisted of a Panoview Ultra endoscope with a 10-mm diameter and 30° head angle (Richard Wolf, catalog no. 8934462), connected to an Alvium 1800 U-508m 5. Imp monochrome camera (Allied Vision, catalog no. 14662) using a RFWO-Zoom Adapter (Richard Wolf, catalog no. 85261504). The camera was controlled via MicroManager (v. 2.0.3) and operated without cooling or gamma adjustment, at an exposure time of 30 ms and with gain set to 48 dB. Illumination from a 470-nm LED (Thorlabs, catalog 30

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[0246] DK17352PC no. M470L5, 810mW) was collimated, filtered through a 469-nm band pass filter with a 35-nm band width (Thorlabs, MF469-35), and focused into a liquid light guide (Richard Wolf, catalog no. 806550231) to the endoscope, with a maximum power density between 2.5 and 8 mW / cm2, depending on the field of view . Emission was filtered with a 520nm band pass filter with a 36-nm band width (Edmund Optics, catalog no. 67-016) attached to the camera adapter. Care was taken to ensure no transmission overlap between the excitation and emission filters, to avoid reflections induced by the illumination source. Images were saved as 8 bit (0 to 255). Proof of principle data with this imaging setup was acquired on a facial nerve of a pig injected with 5 mg / kg of sodium fluorescein (Figure 6).

[0247] Example 8: InVitro Sensitivity Tests with Custom Endoscope and Da Vinci Firefly In vitro sensitivity tests were conducted with 10-pL capillary tubes (Hirschmann, catalog no.

[0248] 9000110) filled with sodium fluorescein at concentrations ranging from 0.5 to 20 pg / ml, diluted in saline. The capillaries were positioned atop fresh excised porcine rib tissue, obtained from a local butcher. Comparative imaging was conducted using the custom endoscope (imaging setup B) and the Da Vinci Firefly Xi system with its integrated 30° endoscope.

[0249] The Da Vinci Xi imaging system was evaluated under two configurations: the optimized setting (maximum Firefly illumination with minimal white light) and the default setting (50% white light and 50% Firefly illumination). To assess the impact of a more specific band-pass filter detection, images were acquired with the custom endoscope paired with Firefly illumination. Subsequent experiments were conducted using the custom endoscope’s illumination system to validate the combined effect of optimized illumination clean-up and band-pass filter imaging (Figure 7).

[0250] To further compare ability of both imaging systems to detect fluorescein buried within tissue, a capillary containing 20 pg / mL sodium fluorescein was embedded approximately 1 mm below porcine tissue. Imaging was performed using the optimized configuration of the Da Vinci Firefly Xi system and the custom endoscope with custom illumination (Figure 8).

[0251] Example 9: Data Processing

[0252] Images were post-processed utilizing Python 3 and Fiji distribution of ImageJ. For all images, 5 or 10 dark frames were acquired for each exposure time and averaged. These averaged dark frames were then used to subtract the dark current from each experimental image. Images were 31

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[0254] DK17352PC then further corrected for exposure time to yield units in counts / s. The displayed linear contrast on each image was optimized and adjusted in Fiji. All images were saved as BMP files on Fiji, then cropped and rearranged as figures on Adobe Illustrator.

[0255] Example 10: Image Analysis

[0256] Weber values for the fluorescence nerve images were calculated to define contrast with the following:

[0257]

[0258] where Is represents the specified region of interest while lb represents the region of background.

[0259] Example 11: Feasibility study

[0260] To evaluate the ability of sodium fluorescein for nerve visualization, we conducted a feasibility clinical trial on the visualization of cranial nerves in patients undergoing salivary gland tumor removal (Figure 2). Seven patients were administered intravenous sodium fluorescein at a dose of 1 mg / kg at anesthesia induction. Using a custom imaging setup (Figure 1) optimized to enhance fluorescence signal while minimizing reflected light, we successfully visualized the facial nerve (CN VII) with significantly greater contrast compared to visible light alone between 2 and 3.5 hours after dye administration (Figure 2). Weber contrast ratios to quantify facial nerve fluorescence compared to background tissues (mean ± SD) was 2.5 ± 1.0. Notably, we were able to highlight small branches of the facial nerve around 1 to 2 mm in size without loss of fluorescence, which were difficult to detect with visible light alone (Figure 2a to 2c). All facial nerve branches were further confirmed intraoperatively via electromyography at 0.5 mA stimulation.

[0261] Example 12: Comparative Examples

[0262] To assess the viability of clinically approved commercial imaging systems compared to our custom system, we conducted a comparative experiment using the Zeiss Kinevo microscope equipped with the Yellow 560 Filter, a commonly utilized system for fluorescein imaging. The Kinevo microscope was able to detect strong nerve fluorescence signals compared to surrounding tissues after further contrast adjustment (Figure 3). However, light reflections which are stronger than the fluorescence signal at this dose of Img / kg made it more challenging to visualize finer nerve branches in comparison to the more optimized setup, even after contrast adjustments (Figure 3e-g).

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[0265] DK17352PC

[0266] Example 13: Application to robotic-assisted surgeries

[0267] To further extend the application of sodium fluorescein imaging to robotic-assisted surgeries, we employed the Da Vinci Xi surgical system, focusing on nerve visualization of the superior hypogastric plexus in a patient undergoing colorectal surgery. Using the 30° endoscope in standard mode, we set the lowest white light and highest Firefly fluorescence intensities to optimize imaging conditions. One patient was injected with a dose of 5 mg / kg sodium fluorescein anesthesia induction to ensure adequate fluorescence signal strength. This setup successfully increased the contrast of pelvic nerves at approximately 1.5 h hours after injection, revealing structures otherwise not visible under white light (Figure 4). Notably, photobleaching from fluorescein was not detected during the surgery under at least 30 minutes of continuous illumination. Additionally, comparative experiments to a customized endoscope system highlight that slight optimization of light source and detection filters can further improve nerve detection sensitivity in minimally invasive settings (Figure 7-8).

[0268] Example 14: Murine pre-clinical model

[0269] To determine feasibility and potential for translation prior to clinical trial enrollment, we also tested our hypothesis to use sodium fluorescein to identify nerves in murine pre-clinical models. Fluorescence from fluorescein administration significantly improved nerve contrast compared to visible light alone (Figure 5). The results from the pan body survey of nerve fluorescence suggest that this nerve identification technique can be applied to any anatomical region or surgery type (Figure 5).

[0270] Example 15

[0271] In both our pre-clinical and clinical data, we noted other structures that also provided fluorescence intraoperatively after the administration of fluorescein. Artifacts from electrocautery demonstrated fluorescence with intensity similar to that of nerves (Figure 3a-b). This cautery artifact, however, was easily distinguished from nerves on white light given the darkened appearance of cautery. Similarly, overlying fascia also showed fluorescence but could be distinguished from nerves by visual inspection. Lymph nodes also provided fluorescence but were easily distinguished from nerves given their differences in structure (Figure 2a). In robotic surgery, the ureters were expectantly fluorescent given the renal excretion clearance pattern of fluorescein (Figure 4c), which can be used to avoid ureteral injury.

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[0274] DK17352PC

[0275] Our study demonstrates that high-contrast imaging of fluorescein-labeled nerves is possible intraoperatively with clinical imaging systems, particularly in robotic surgery using the Da Vinci Firefly system. With our custom imaging systems that minimized reflections, we were not only able to highlight peripheral nerves, but also highlight small branches not seen with visible light. Thus, adjustments in commercially available imaging systems, such as optimizing fluorescence filters to remove reflections, have the potential to remarkably enhance nerve contrast in open and robotic-assisted procedures.

[0276] Collectively, our results indicate that sodium fluorescein can highlight nerves compared to visible light for intraoperative nerve identification. In this study, we have seen benefit in identifying small nerve branches and nerves hidden under fat not visible under white light so we reason that select surgeries that have high rates of nerve injury will have the greatest benefit.

[0277] Example 16: Rectal resection imaging (Fig. 9)

[0278] Fluorescence imaging was performed during a rectal resection using a custom fluorescence-enabled endoscope as specified herein above, following systemic administration of sodium fluorescein at 5 mg / kg. In this human surgical case, nerve structures within the operative field were visualized using the custom endoscope. The custom endoscope provided clearer nerve visualization, which reduced reflected light and background signal. These findings demonstrate improved nerve visualization by the method and device described herein in a human surgical setting.

[0279] Example 17: Acute and chronic nerve injury models in mice (Fig. 10)

[0280] To assess whether sodium fluorescein-based nerve fluorescence persists in the setting of nerve injury or degeneration, two murine nerve injury models were evaluated. In an acute injury model, the sciatic nerve was subjected to a controlled crush injury 24 h prior to fluorescence imaging. The mice were imaged 5 hours after intravenous administration of 15 nmol / gram sodium fluorescein . Imaging demonstrated a localized loss of fluorescence signal at the site of injury, while adjacent, uninjured portions of the nerve continued to exhibit fluorescence, allowing clear differentiation between injured and intact nerve segments (Fig. 10a).

[0281] In a chronic injury model, a portion of the facial nerve was surgically transected and allowed to degenerate over time. At 12 weeks post-transection, distal nerve segments exhibited marked 34

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[0283] DK17352PC atrophy and were difficult to distinguish using visible light alone. Following sodium fluorescein administration (15 nmol / g, imaging at 5 h after iv injection), these atrophied distal nerve segments were visualized by fluorescence imaging, indicating retention of detectable signal in chronically injured and degenerating nerves. Together, these findings demonstrate that sodium fluorescein enables visualization of both acutely injured and chronically degenerated nerves, supporting its applicability for identifying severed nerves in reinnervation or reconstructive procedures (Fig. 10b).

[0284] Example 18: Clinical imaging of chronic nerve injury (Fig. 11)

[0285] The feasibility of fluorescence-based visualization of chronically injured nerves was further evaluated in a patient imaged one year after facial nerve transection due to tumor involvement and subsequent adjuvant chemoradiation. During a facial nerve reanimation procedure, 1 mg / kg sodium fluorescein was administered systemically, and intraoperative visible and fluorescence images were acquired. Despite a re-operated surgical field characterized by scarring, distorted fascial planes, and radiation-associated fibrosis, fluorescence imaging enabled identification of the distal trunk and atrophied branches of the facial nerve. Several nerve branches that were poorly discernible or not identifiable under visible light alone exhibited detectable fluorescence signal.

[0286] Example 19: Histopathology and microscopic localization (Fig. 12)

[0287] Histological cross-sections of an unfixed porcine facial nerve following sodium fluorescein administration demonstrated accumulation of fluorescence within nerve-associated tissue. Confocal and two-photon fluorescence microscopy combined with second harmonic generation revealed localization of fluorescein within the collagen-rich epineurium surrounding the nerve, as visualized by second harmonic signal from collagen structures.

[0288] Example 20: Further improvements to the custom endoscope design

[0289] In addition to the endoscope system described herein above, we designed a new hardware and software architecture to enable “multi-modal” visualization using a single color camera in combination with two independently controlled illumination sources. The system alternates between a broadband white light source and a blue laser excitation source (488 nm laser with a BP488-3 clean-up filter), which are temporally triggered and synchronized with image acquisition. Each illumination mode provides complementary information (white light / reflectance for general guidance and fluorescence for increased contrast) and is acquired 35

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[0291] DK17352PC independently, allowing the surgeon to selectively display white-light images, fluorescence images, or a combined overlay, according to user preference. The switching between illumination modes occurs at video frame rates such that no perceptible flickering is observed by the surgeon during intraoperative use. The system uses a notch-pass filter on the detection side to suppress reflected laser excitation light while preserving detection of most of the visible spectrum, including fluorescein emission.

[0292] References

[0293] 1. Antoniadis, G., et al. Iatrogenic nerve injuries: prevalence, diagnosis and treatment. Dtsch Arztebl Int 111, 273-279 (2014).

[0294] 2. Paun, B.C., Cassie, S., MacLean, A.R., Dixon, E. & Buie, W.D. Postoperative complications following surgery for rectal cancer. Ann Surg 251, 807-818 (2010).

[0295] 3. Prim, M.P., De Diego, J.I., Verdaguer, J.M., Sastre, N. & Rabanal, I. Neurological complications following functional neck dissection. Eur Arch Otorhinolaryngol 263, 473-476 (2006).

[0296] 4. Kyriazis, I., et al. Different Nerve-Sparing Techniques during Radical Prostatectomy and Their Impact on Functional Outcomes. Cancers (Basel) 14(2022).

[0297] 5. Holland, N.R. Intraoperative electromyography. J Clin Neurophysiol 19, 444-453 (2002).

[0298] 6. Wang, L.G., et al. Near-infrared nerve-binding fluorophores for buried nerve tissue imaging. Sci TranslMed 12(2020).

[0299] 7. Hingorani, D.V., et al. Nerve-targeted probes for fluorescence-guided intraoperative imaging. Theranostics 8, 4226-4237 (2018).

[0300] 8. Nazzi, V., et al. Assessing the role of sodium fluorescein in peripheral nerve sheath tumors and mimicking lesions surgery: An update after 142 cases. Front Oncol 12, 1070878 (2022).

[0301] 9. Wolfe, D.R. Fluorescein angiography basic science and engineering. Ophthalmology 93, 1617-1620 (1986).

[0302] 10. Zhang et al. (2024), JNeurosurg Sci 67(3):374

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Claims

1. Attorney Ref.: 097147-0181DK17352PC Claims1. A method for visualizing an anatomic structure in an operating zone of a subject during a surgical procedure comprising:(i) contacting said anatomic structure with a fluorescein dye;(ii) illuminating said operating zone with light exciting said fluorescein dye; and (iii) thereby visualizing said anatomic structure,wherein said anatomic structure is a nerve structure, and / or a fascia.

2. The method of claim 1, wherein said visualizing comprises administering said fluorescein dye to said subject at a dose of from 0.1 mg / kg body weight to 50 mg / kg body weight, preferably at a dose of from 0.25 mg / kg body weight to 25 mg / kg body weight, still more preferably at a dose of from 0.5 mg / kg body weight to 12.5 mg / kg body weight, even more preferably at a dose of from 2 mg / kg body weight to 10 mg / kg body weight, most preferably at a dose of from 1 mg / kg body weight to 5 mg / kg body weight, wherein said fluorescein dye preferably is administered systemically.

3. The method of claim 1, wherein said visualizing comprises systemically administering said fluorescein dye at least 30 min before visualizing, preferably at least 60 min before visualizing, and / or wherein said visualizing comprises administering said fluorescein dye at most 10 hours before visualization, preferably at most 7.5 hours before visualizing, still more preferably at most 5 hours before visualizing, most preferably at most 3 hours before visualizing.

4. The method of claim 1, wherein said visualizing comprises illuminating said operating zone with radiation having a wavelength in the range of from 400 nm to 500 nm, preferably of from 420 nm to 495 nm, more preferably of from 425 to 495 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 451 nm to 487 nm and / or wherein said visualizing comprises detecting light emitted from the operation zone at a wavelength of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm.

5. The method of claim 1, wherein said visualizing comprises identifying at least one anatomic structure within said operating zone.374917-8732-8394.1Attorney Ref.: 097147-0181DK17352PC6. The method of claim 1, wherein said anatomic structure is a nerve structure and wherein said surgical procedure is or comprises a salivary gland surgery, colorectal surgery, prostate surgery, gynecological surgery, hip replacement surgery, thyroid surgery, and / or ear surgery.

7. The method of claim 1, wherein said surgical procedure is performed at least partially by an electronic and / or semi-automated or automated equipment, preferably by a robotic surgical system.

8. The method of claim 1, wherein damaging said anatomic structure visualized is avoided.

9. The method of claim 1, wherein said anatomic structure is a nerve structure and wherein said nerve structure is a target of a neurectomy, preferably wherein said surgical procedure is vagotomy, presacral neurectomy, vestibular neurectomy, neurectomy to alleviate nerve entrapment, intercostal cutaneous nerve neurectomy, or lateral femoral cutaneous nerve neurectomy.

10. The method of claim 1, wherein said fluorescein dye is fluorescein (CAS No. 2321-07- 5 and / or CAS No. 518-45-6) or a salt thereof, preferably sodium fluorescein (CAS No.518-47-8) or potassium fluorescein (CAS NO. 6417-85-2).

11. A method for treating a subject by a surgical procedure comprising:(A) visualizing an anatomic structure in an operating zone of said subject during said surgical procedure according to the method according to claim 1; and(B) thereby treating said subject by said surgical procedure.

12. A surgical detection device comprising an illumination unit adapted for at least partially illuminating an operating zone of a subject with radiation exciting a fluorescein dye.

13. The surgical detection device of claim 12, wherein said illumination unit comprises (i) a radiation source emitting radiation having a wavelength in the range of from 400 nm to 550 nm, preferably of from 420 nm to 530 nm, more preferably of from 425 to 500 nm, even more preferably of from about 450 nm to 490 nm, most preferably of from 384917-8732-8394.1Attorney Ref.: 097147-0181DK17352PC 451 nm to 487 nm; and, optionally,(ii) a short-pass filter with a cutoff wavelength in the range 480 nm to 495 nm, and / or (iii) a long-pass filter with a cutoff wavelength in the range of from 400 nm to 450 nm; and / or(iv) a band-pass filter with a central wavelength in the range of from 450 nm to 490 nm and with a bandwidth in the range of from 50 nm to 10 nm;wherein said filters (ii) to (iv), if present, are arranged to intervene between the radiation source and the operating zone.

14. The surgical detection device of claim 12, wherein said surgical device further comprises a detector unit comprising(aa) a detector adapted for detecting radiation with a wavelength in the range of from 475 nm to 600 nm, preferably of from 500 nm to 550 nm; and, optionally,(bb) a long-pass filter with a cutoff wavelength in the range of from 490 nm to 500 nm; (cc) a short pass filter with a cutoff wavelength in the range of from 540 to 600 nm; and / or(dd) a band-pass filter with a central wavelength in the range of from 520 nm to 550 nm and with a bandwidth of from 50 nm to 10 nm;wherein said filters (bb) to (dd), if present, are arranged to intervene between the operating zone and the detector unit.

15. The surgical detection device of claim 14, wherein said illumination unit is configured to provide pulsed illumination of the operating zone, wherein said pulsed illumination is synchronized with said detecting radiation, in a preferred embodiment wherein said pulsed illumination and said detecting radiation are performed at a rate of video capture, preferably of from 10Hz to 100Hz.

16. A surgical system comprising (a) a surgical detection device according to claim 12 and (B) a robotic surgical device, wherein the surgical detection device preferably is adapted for automatically detecting at least one anatomic structure in an operating zone, and wherein said robotic surgical device preferably is adapted to avoid damaging the anatomic structure detected by said surgical detection device.

17. A kit for visualizing an anatomic structure in an operating zone during a surgical 394917-8732-8394.1Attorney Ref.: 097147-0181DK17352PC procedure, the kit comprising (AA) a fluorescein dye and (BB) a surgical detection device according to claim 12; and / or a manual comprising instructions for performing a method according to any of the preceding claims relating to a method.

18. A method of histopathologically visualizing an anatomic structure rich in connective tissue, comprising:(I) contacting said anatomic structure with a fluorescein dye,(II) illuminating said anatomic structure with light exciting said fluorescein dye; and (III) thereby visualizing said anatomic structure.404917-8732-8394.1

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