WO2024223501A1

WO2024223501A1 - A reference device and a method of manufacturing thereof

Info

Publication number: WO2024223501A1
Application number: PCT/EP2024/060970
Authority: WO
Inventors: Stefan HARMSEN; Bonifacius Willem Alexander DE JONGE
Original assignee: Absofi Bv
Priority date: 2023-04-26
Filing date: 2024-04-22
Publication date: 2024-10-31

Abstract

A reference device for calibrating fluorescence imaging systems in fluorescence-guided medical interventions and method for manufacturing said reference device wherein the reference device comprises at least one reference area (2) comprising at least one fluorescent contrast agent capable of fluorescing when stimulated by electromagnetic waves, wherein the at least one fluorescent contrast agent is inkjetprinted on a surface of the reference device and configured to exhibit a plurality of fluorescence intensities.

Description

A reference device and a method of manufacturing thereof

The invention relates to the field of fluorescence imaging systems, particularly to a reference device for calibrating fluorescence imaging systems in fluorescence-guided medical interventions and aid in clinical decision-making and a method of manufacturing thereof.

An important field of application of contrast measurements is the field of medical imaging. In particular, the field of fluorescence- and/or molecular imaging in which non-targeted contrast agents such as indocyanine green (ICG) - a near-infrared fluorescent (NIRF) dye - or tissue-agnostic contrast agents such as for example pafolacianine - a folate receptor-targeted NIRF dye - are used to provide contrast to tissue of interest (e.g., tumor, nerve, vasculature, ureter, bile ducts, etc.) from other surrounding tissues in a diagnostic- or therapeutic clinical setting. Within a certain timeframe following administration (e.g., intravenous, topical, oral, sublingual, parenteral, interstitial, etc.) such contrast agents accumulate in the tissue of interest and can be visualized by specialized medical imaging systems. Typically, such medical imaging systems include an excitation source such as a laser or LED that is capable of exciting the contrast agent of interest to irradiate the tissue and/or sample containing said contrast agent, and, a camera consisting of an image sensor to detect the fluorescence that is produced by the contrast agent upon irradiation by the excitation source (the camera may also include an emission filter to block the detection from radiation produced by the excitation source). The fluorescence signal intensity of the tissue of interest can be used but is not limited to determining the perfusion state of the tissue, the location and extent of a benign and/or malignant tumor, the location and extent of critical tissues such as nerves, ureters, lymph nodes, etc. in a variety of clinical scenarios, i.e. especially when ambient measurement parameters are subject to variation. It is common practice to use an external fluorescence reference when performing the measurements. The reference device is placed within the same field of view (FOV) as the tissue of interest (preferably in the same plane) and, when irradiated, the calibration device emits a constant and defined fluorescence signal which is recorded together with the fluorescence signal of the tissue of interest. The use of a fluorescence reference device allows to directly compare different measurements and to normalize measurement results based on the defined signal intensity of the fluorescence standard. It further allows compensating changes in the measurement conditions during a measurement (such as change in intensity of ambient light, change of exposure parameters of the camera, or change of parameters of the radiation)and between measurements on different systems, for instance during a multicenter clinical trial.

For reference purposes as described above, pure dye contrast agent such as ICG is usually dissolved in water or methanol immediately before use. With this liquid sample serving as the fluorescence standard a fluorescence measurement can be performed as described above. Since contrast agents such as ICG are not stable when exposed to air humidity and light, the reference device must be prepared immediately before use, which is a major disadvantage when used during an urgent medical procedure. Furthermore, the practice of preparing reference devices is time-consuming and owing to the instability of such liquid reference samples, fresh samples are preferably prepared before each use case, which introduces variability between cases due to errors associated with preparation of the liquid fluorescence standard samples. Moreover, the absorption and fluorescence properties of such pure dissolved contrast agent standard samples are not equal to the properties of the contrast agent, which may vary due to difference in polarity between the media and/or association of the contrast agent with plasma proteins such as albumin that will shift the absorption maximum by several nanometers.

It is not possible to use pure dry powder of ICG or other (optical) contrast agents (e.g., protoporphyrin IX (PPIX), AF488, bodipy, fluorescein, methylene blue, oxazines, cy5, cy7, cy7.5, IRdye700DX, BM-104, ASP5354, JAS239, cypate, S0456, ZW800-1 , IRdye800CW, IRdye800BK, DY800) or derivatives thereof, for reference purposes as described above, since pure contrast agent powder also exhibits absorption and fluorescence properties different from the dissolved contrast agent. Furthermore, contrast agents like ICG may alter their properties due to ambient conditions such as humidity. US2005/0219524 discloses the use of a calibration aid incorporating a fleece carrier sheet (preferably with a porosity of above 80%) as a substrate wherein said substrate is impregnated with albumin-bound ICG, which is placed on top of a white paper backing sheet. The calibration aid can be placed at a distance from the tissue of interest such that both can be irradiated simultaneously by an excitation source and measured simultaneously by a medical imaging system as described above. However, there is concern over deterioration of fluorescence characteristics caused by denaturation of protein, since the contrast agent is combined with albumin protein in the calibration aid. Furthermore, since only one side can be imaged for each measurement, the fluorescence intensity of the tissue of interest can only be compared to a single fluorescence intensity level per measurement. Since the response of the camera sensors typically is not linear across a range of fluorescence intensities associated with different clinically-relevant concentrations of the contrast agent, the use of a calibration aid with a single fluorescence intensity level precludes the estimation of contrast agent concentrations in the tissue of interest when the fluorescence intensity of the tissue of interest is not equal to the calibration aid during the fluorescence measurement of the camera system.

It is necessary to standardize the imaging conditions to evaluate perfusion objectively and reproducibly. Indocyanine green fluorescence has not been clearly demonstrated to reduce anastomotic complications, but most prior art studies have been based on the qualitative evaluation of ICG fluorescence in the colon wall or mucosa. It is possible that ICG angiography has not been verified as a predictor of anastomotic complications because qualitative evaluation alone is limited in accurately distinguishing changes in the microcirculation of the large intestine. Quantitative blood flow analysis is necessary to measure changes in colonic microcirculation to predict bowel viability.

Due to the above-described reasons, it is difficult to (semi-)quantitatively assess the concentration of contrast agent in a tissue of interest using such calibration means. And, despite advances in camera systems and displays, interpretation of the fluorescence signal is not standardized and thus remains up to the surgical operator and is therefore prone to interpretation variability. It is an object of the current invention to correct the short-comings of the prior art and to provide a fluorescence reference device that obviates the need for protein inclusion in order to increase its shelf-life, wherein the manufacturing of such a reference device is highly scalable. This and other objects which will become apparent from the following disclosure, are provided with a reference device and a method of manufacturing thereof having the features of one or more of the appended claims.

In a first aspect of the invention, the reference device for calibrating fluorescence imaging systems in fluorescence-guided medical interventions comprises at least one reference area comprising at least one fluorescent contrast agent capable of fluorescing when stimulated by electromagnetic waves, wherein at least one fluorescent contrast agent is printed on a surface of the reference device and configured to exhibit a plurality of fluorescence intensities.

The at least one fluorescent contrast agent preferably comprises a fluorescent dye wherein the fluorescence intensities are controlled by concentration levels of said fluorescent dye inside the at least one fluorescent contrast agent.

The fluorescent dye is preferably capable of emitting light with a wavelength or wavelengths between 400 nm and 1800 nm when stimulated by electromagnetic waves.

More particularly, the fluorescent dye is preferably a near-infrared fluorescent dye capable of emitting light with a wavelength or wavelengths between 600 nm and 950 nm when stimulated by electromagnetic waves.

The concentration levels of the fluorescent dye inside the at least one fluorescent contrast agent are preferably between 0.001 nanomolar and 1000 micromolar.

The at least one fluorescent contrast agent preferably comprises a solvent-based transparent ink compatible with inkjet printers and pad printers. The solvent can be water, alcohol, formaldehydes, dimethylformamide, n-methylpyrrolidone or dimethylsulfoxide or a similar solvent. The at least one fluorescent contrast agent preferably comprises a waxy resin-based ink compatible with solid ink printers.

The at least one fluorescent contrast agent preferably comprises a granulated ink compatible with toner printers.

The fluorescent dye preferably comprises a derivative of at least one group of cyanines, xanthenes, triarylmethanes, (na)phthalocyanines,(proto)porphyrins, boron- dipyrromethenes, squaraines, croconaines, oxazines, polymethines, chromenylium, (chalcogeno)pyrylium, and flavylium.

The fluorescent dye preferably comprises one of indocyanine green, methylene blue, protoporphyrin IX disodium, BM-104, cy5, cy5.5, cy7, cy7.5, ZW800-1 , asp5354, cypate, DY800, IRdye800CW, IRdye700DX, S0456, ADS680HO and ADS680WS.

The fluorescent dye preferably comprises an optically-active nanoparticle such as a quantum dot, a dye-embedded silica nanoparticles, or a gold nanocluster, with a particle size between 1 nm and 1000 nm.

The at least one fluorescent contrast agent preferably comprises a solvent such as alcohol, dimethylformamide, n-methylpyrrolidone or dimethylsulfoxide.

The calibration device preferably comprises a substrate comprising at least one of paper, plastic, PVC, cellulose, cellulose acetate, textile or metallic material.

The reference device preferably comprises a graduated ruler or scale for measuring a length.

The reference device preferably comprises at least one identification label for identifying the fluorescent contrast agent.

The reference device can be used for providing ground truth fluorescence values when training an artificial neural network. In some embodiments, the reference device can serve as the ground truth to train a machine learning (ML) algorithm or artificial intelligence (Al) engine. “Ground truth” is a term commonly used in the fields of statistics, machine learning and artificial intelligence. It refers to the correct or “true” answer to a specific problem or question. It is a “gold standard” that can be used to compare and evaluate model results. For example, some envisioned applications relate to ML algorithms or Al engines providing “true” concentrations of (near-infrared) fluorescence agent present in tissue of interest that is located beneath other tissues and/or is covered by other tissues and/or is embedded within other tissues following administration of said contrast agent. As a result of said contrast agent being located at a certain depth, the fluorescence intensity of said agent is decreased relative to the same agent being imaged superficially. If the depth of the tissue or structure wherein said contrast agent is known, the reference device can be used in combination with an ML algorithm or Al engine to calculate the true concentration of the contrast agent in the embedded tissue.

In some other embodiment, the reference device can also be used in providing ground truths for specific clinical applications where fluorescent intensity values differ. By first empirically matching fluorescent intensity values to disease or disease status, the correct concentration ranges for specific applications are derived. These are then embodied onto the reference device, as this allows for custom concentration ranges. The specific concentrations range on the reference device can then be used in conjunction with fluorescent images of said applications. These image sets are then labelled as ground truths for an AI/ML model to be trained on.

In a second aspect of the invention, the method for manufacturing a calibration device comprises the steps of: providing a substrate comprising at least one of paper, plastic, PVC, cellulose, cellulose acetate, textile or metallic material; and creating at least one reference area on said substrate by: preparing at least one fluorescent contrast agent by dissolving a fluorescent dye in a solvent mixed with a transparent printer-ink, wherein a concentration level of the fluorescent dye inside the at least one fluorescent contrast agent is between 0.001 nanomolar and 1000 micromolar; and printing said at least one fluorescent contrast agent on the substrate.

The solvent and the transparent printer-ink are preferably compatible with inkjet printers or with toner printers or with solid ink printers or with solid ink printers.

The method preferably comprises the step of providing the fluorescent dye as a nearinfrared fluorescent dye.

The method preferably comprises the step of providing the transparent ink as a waterbased ink.

The method preferably comprises the step of providing the fluorescent dye as a derivative of at least one group of cyanines, xanthenes, triarylmethanes, (proto)porhyrines, (na)phthalocyanines, boron-dipyrromethenes, squaraines, croconaines, polymethines, oxazines, (chalcogeno)pyrylium, chromenylium, and flavylium.

The method preferably comprises the step of providing the fluorescent dye as one of indocyanine green, methylene blue, cy5, cy5.5, cy7, cy7.5, BM-104, protoporphyrin IX disodium, S0456, IRdye800CW, lrdye700DX, ZW800-1 , cypate, DY-800, ASP5354, ADS680HO and ADS680WS, or a derivative thereof.

The method preferably comprises the step of providing the solvent as one of alcohol, dimethylformamide, n-methylpyrrolidone or dimethylsulfoxide.

The method preferably comprises the step of providing an optically-active nanoparticle such as a quantum dot, a dye-embedded silica nanoparticles, or gold nanocluster, with a size between 1 nm and 1000 nm.

The step of printing the at least one fluorescent contrast agent on the substrate using an inkjet printer preferably comprises the step of filling a first ink cartridge of said inkjet printer with a first fluorescent contrast agent comprising a first concentration level of a first fluorescent dye and filling at least a second ink cartridge with at least a second fluorescent contrast agent comprising at least a second concentration level of at least a second fluorescent dye.

The first fluorescent dye and the at least second fluorescent dye are preferably the same.

The first concentration level and the at least second concentration level are preferably equal.

The step of printing the fluorescent contrast agent on the substrate using an inkjet printer preferably comprises the step of filling at least one ink cartridge exclusively with a transparent ink.

The method preferably comprises the steps of: creating a computer-readable image by correlating red, green, black (RGB) values of the computer readable image to the cyan, magenta, yellow, key (CMYK) values of the inkjet printer; and using said computer-readable image to print the at least one contrast agent on the substrate.

The method preferably comprises the step of controlling the fluorescence intensities of the at least one contrast agent by controlling color transparency levels in the computer-readable image.

The method preferably comprises the step of calibrating the reference device by:

- creating a plurality of serums (6) by dissolving the fluorescent dye in a solvent containing between 1 % and 8% weight of albumin, wherein the concentration levels of the fluorescent dye are between 0.001 nanomolar and 1000 micromolar;

- comparing the fluorescent intensities in the at least one reference area of the reference device to the fluorescent intensities in the plurality of serums (6) for associating fluorescent intensities in the at least one reference area of reference device to the fluorescent intensities in the plurality of serums (6). The method preferably comprises the step of providing graduated rulers or scales on the reference device for measuring a length and/or resolution.

The method preferably comprises the step of providing at least one identification label on the reference device for identifying the fluorescent contrast agent.

The method preferably comprises the step of laminating the substrate using a laminating pouch after printing the at least one fluorescent contrast agent on the substrate.

The method preferably comprises the step of fixing the at least one fluorescent contrast agent using a fixative agent, such as aldehydes, after printing the at least one fluorescent contrast agent on the substrate.

The method preferably comprises the step of sterilizing the reference device for medical use. In some embodiments, the laminated reference device is sterilized and enclosed in packaging material to keep it sterile before actual use as a disposable. Such a sterile reference device can be held over or placed in or close to the sterile surgical field and imaged in situ within the same field-of-view (FOV) using a medical imaging device to obtain an accurate estimation of the (near-infrared) contrast agent present in the tissue of interest following administration of said contrast agent. In another embodiment, the sterilized reference device can be placed close to freshly excised tissue of interest on a back-table in the surgical suite and imaged ex vivo within the same field-of-view (FOV) using a medical imaging device to obtain an accurate estimation of the (near-infrared) contrast agent present in the excised tissue of interest following administration of said contrast agent. The reference device is intended for single use.

The invention will hereinafter be further elucidated with reference to the drawing of an exemplary embodiment of a reference device according to the invention that is not limiting as to the appended claims.

In the drawing: figure 1 shows the reference device of the invention in a side view; figure 2 shows the reference device of the invention in a front view from above; figure 3 shows the reference device and a serum of the invention in a front view from above. figure 4 shows the correlation of the reference device in terms of fluorescence intensity relative to the same contrast agent dissolved in 1 % albumin. figure 5 is a flow diagram illustrating aspects of a method of generating a fluorophore concentration map. figure 6 is a flow diagram illustrating aspects of a method of determining intensity information of patches. figure 7 is a flow diagram illustrating aspects of a method of fitting a data model to the intensity information of patches.

The manufacturing of the reference device of the invention comprises four stages: Ink formulation;

Printer configuration;

Inkjet-mediated contrast agent titration; and Validation.

Ink formulation

First, the inks for each (near-infrared) fluorescent contrast agent are prepared by dissolving a fluorescent dye e.g. indocyanine green, methylene blue, protoporphyrin IX disodium, S0456, ADS680HO, ADS680WS, IRdye800CW, IRdye700DX or any other contrast agent or derivative thereof from the following, but not limited to the groups of cyanines, xanthenes, triarylmethanes, (na)phthalocyanines, (proto)porphyrines, (chalcogeno)pyrylium, oxazines, boron-dipyrromethenes, squaraines, croconaines, polymethines, chromenylium, flavylium, etc. in dimethylsulfoxide (DMSO), or any other polar solvents such as alcohols, dimethylformamide (DMF), or n-methylpyrrolidone (NMP) at a concentration of 100 millimolar or any other appropriate concentration. These stock solutions can be further diluted into a water-based inkjet-compatible transparent ink (e.g. Epson Gloss Optimizer (T3240), Canon Chroma Optimizer (PFI-300CO), etc.) at a concentration between 1 nanomolar - 10 millimolar (depending on the fluorescent dye).

Printer configuration An inkjet printer can be used for printing by replacing the original ink cartridges by refillable cartridges containing the (near-infrared) fluorescent inks; for example: the yellow (Y) cartridge contained 14 mL 50 micromolar ICG in transparent ink (e.g. T3240 for Epson SC-P400), the magenta (M) contained 14 mL 500 micromolar PPIX disodium in in transparent ink (e.g. T3240 for Epson SC-P400 for Epson SC-P400), and the cyan (C) contained 14 mL 5 micromolar ADS680WS in in transparent ink (e.g. T3240 for Epson SC-P400). In order to allow for adjusting the color transparency, other cartridges can be replaced with transparent ink (e.g. T3240 for Epson SC-P400).

Inkjet-mediated contrast agent titration

The amount of the formulated inks that were dispensed from the printer can be controlled by standard image-processing software (e.g. MS powerpoint, photoshop, inkscape, scribus, GIMP, etc.). Typically, PowerPoint was used for standard design (see below). The design constituted a ruler (metric scale) and a number of square fields (n>1 ). While the printer utilizes a CMYK colorspace, Powerpoint utilizes an RGB color space, therefore values of red (R), green (G), and blue (B) in Powerpoint can be set to approximate cyan (C), magenta (M), or yellow (Y). For example, to exclusively print from the ICG-containing yellow cartridge, all the fields can be filled with the setting that closely approximated yellow in the CMYK colorspace; R: 255, G: >238, B: 0. Moreover, to dispense different amounts (i.e. concentrations) of ICG, transparency levels can be set anywhere from 0-100%. Next, the design can be saved as a “name”. PDF and imported in Scribus - an open-source image-processing tool - to convert the RGB colorspace to CMYK colorspace and saved as “name(converted)”.PDF again. The standard design (“name(converted)”.pdf) can then be printed on the substrate (e.g. white, transparent, or colored PVC, cellulose, cellulose acetate, plastic, paper, metal, textile, fabric, etc.) of a certain shape/dimension (A6,A4,A3,CR80, etc.) using the inkjet printer. Once dried, the substrate can be laminated using a laminator/laminating pouch, or fixed using a fixative.

The reference areas can be printed in such a way that a first reference area comprising a first fluorescent dye is a series of discretely separated areas wherein each area has a different concentration level of this first fluorescent dye, see figure 1 . Alternatively, this first reference area can be printed as a gradient of varying concentrations of the first fluorescent dye. When multiple reference areas are needed, the same process is repeated for the subsequent reference areas, see figure 2.

Validation

The fluorescence intensity of the different reference areas of the reference device are calibrated to pre-defined concentrations of the contrast agent in blood plasma. To achieve this, a serum is prepared by dissolving known concentrations of the contrast agents such as ICG in water containing albumin (1 -8% w/v). Next, the inkjet-printed reference device is imaged in the same field of view (FOV) as the known ICG-albumin concentrations. The fluorescence intensities of the reference areas are compared/calibrated to the known “blood plasma concentrations” in the serum.

If there is a mismatch, transparency levels of the fields on the cards can be recalibrated to match fluorescence intensity of known ICG concentrations in the serum like it is shown in figure 3.

Instead of water containing albumin, any suitable solvent (composition) may be used that simulates the fluorescence properties of the contrast agent in blood plasma. For example, the pre-defined concentrations of the contrast agent may be dissolved in real blood plasma.

Alternatively, in place of the water containing albumin, any suitable composition may be used to simulate the fluorescence properties of any other desired particular body fluid, such as bile, lymph fluid, or cerebrospinal fluid, or of a tissue, for example a fatty tissue. After the calibration, the reference device can be used to assess dye concentrations in the respective body fluid. Any suitable composition may be used that influences the fluorescence properties of an agent to simulate its behavior in a body fluid or tissue.

Yet alternatively, the fluorescence intensity of the different reference areas of the reference device may be calibrated to pre-defined concentrations of the contrast agent in any particular body fluid, such as blood plasma, bile, lymph fluid, or cerebrospinal fluid. The pre-defined concentrations of the contrast agent may be dissolved in samples of the respective body fluid. After such calibration, the reference device can be used to assess dye concentrations in the respective body fluid. Fluorophore concentration assessment

The calibrated reference device can be used to determine the concentration of the fluorescent dye in the particular body fluid that the reference device was calibrated to. This assessment can be done by imaging a relevant body part of a patient and the reference device with a fluorescence imaging system, and comparing the detected fluorescence intensity of the body part to the detected fluorescence intensities of the reference areas of the reference device.

Examples

In certain embodiments, the step of printing the at least one fluorescent contrast agent on the substrate comprises the step of filling a first ink cartridge of said printer with a first fluorescent contrast agent comprising a first concentration level of a first fluorescent dye and filling at least a second ink cartridge of said printer with at least a second fluorescent contrast agent comprising at least a second concentration level of at least a second fluorescent dye.

The cartridges of the printer may be compartments filled with ink that can be installed in the printer simultaneously. This way, the different dyes from different cartridges may be printed on a single substrate in one printing session. For example, existing color cartridges of the printer such as ‘cyan’, ‘magenta’, and ‘yellow’ may be filled with different contrast agents instead of the color ink.

Fluorescence intensities of each fluorescent dye printed on the substrate may be controlled by controlling the size and density of the dots of contrast agent created by the printer on the substrate. This way, the amount of the contrast agent per surface area of the substrate can be controlled to create the different fluorescence intensities.

Specifically, controlling the printer may comprise providing control signals associated with a spot color channel of the printer, to control the amount of the contrast agent that is dispersed from each cartridge onto the substrate per surface area of the substrate. This in turn controls the fluorescence intensity of each fluorescent dye formed on the substrate. The first fluorescent dye and the at least second fluorescent dye may be different fluorescent dyes. For example, different types of dye may be provided in different cartridges of the printer. Further, different concentrations of the same or a different fluorescent dye may be provided in the different compartments to make it easier to print a large range of fluorescence intensities.

The contents of multiple reference devices can be printed side by side on a single substrate. The method may comprise cutting the substrate to form a plurality of reference devices after the printing is completed.

Fluorescence imaging system

A fluorescence imaging system (FIS), as referred to herein, may be a an image capturing system (ICS) for capturing fluorescence images. A fluorophore concentration reference target, which may be referred to hereinafter as ‘target’, may be a calibration reference device containing one or more reference patches of calibrated fluorophore densities, as described hereinabove. A ‘patch’, as referred to hereinafter, may refer to a particular area on a reference target with a uniform fluorophore density that equates the fluorescence intensity of a known fluorophore concentration.

In the following, methods and systems of operating a fluorescence imaging system will be described. These methods and systems may provide a standard for quantifying clinical fluorescence signals and may improve the reproducibility of results across different imaging systems and clinical working groups. A visual representation of tissue(s) of a subject may be generated that is more accurate in terms of data representation, as well as more intuitive for clinicians to use for their clinical decision making. The methods and systems, and the generated visual representation of tissue, may be applicable to various types of tissue (e.g. a variety of pathologies including cancer tumors, bum wounds, and pressure ulcers), and may provide a framework for automatically classifying the tissue (e.g. healthy tissue versus tumor tissue) and/or predicting clinical outcomes (e.g. wound healing timeline).

Conventional medical imaging devices, such as fluorescence imaging systems, have restrictions in accurately assessing fluorophore concentrations. For instance, when visually evaluating fluorescence images that represent the accumulation of a fluorophore in a lymph node, a clinicians’ assessment is confounded by parameters (e.g., brightness, image contrast, image noise) that are independent of properties of the tissue. Additionally, clinicians’ mere visual evaluation of the images is subjective and may vary from clinician to clinician, patient to patient, imaging device to imaging device, and/or imaging session to imaging session.

The methods and systems described herein are useful for characterizing tissue, predicting clinical data or outcomes and presenting image data to the user in a manner that enables more effective clinical decision making to further facilitate predicting clinical outcomes. In particular, the fluorophore concentration map (e.g., concentration image), generated in accordance with the methods described herein (e.g., 500 in Fig 5) for a subject (e.g., a patient) undergoing or having undergone medical imaging, may be a spatial map that concisely shows differences in fluorophore concentrations between image elements (such as, for example, pixels of a two-dimensional image or voxels of a three-dimensional image), expressing differences in clinically relevant attributes. In the following description, when pixels are described, this may be replaced by voxels as appropriate. In some implementations, the fluorophore concentration map may be a visualization of how areas in the imaged subject tissue vary in healing status, developmental stage of a tumor’s growth, and/or other tissue conditions. For example, the fluorophore concentration map may visualize inflammation, disease, or other abnormality of tissue in a way that is easily perceptible and identifiable by a human being. As further described herein, these generated visualizations reduce ambiguity and the effect of a clinician’s subjectivity, by facilitating a standardized protocol for assessing images and providing a way to compare and track assessments of a subject over time across multiple imaging sessions. Thus, these visualizations enable a clinician to make more consistent clinical assessments and/or medical treatment decisions.

Assessment of fluorescence signals in images of tissue is dependent upon the properties of the image capturing system. Due to variability across fluorescence imaging systems, manufacturers, and operating procedures, it is difficult to achieve accurate assessment of fluorescence images across large patient groups, both in research and clinical care settings. The systems and methods for obtaining fluorophore concentration maps according to the present embodiments automatically and accurately determine (an approximation of) the absolute concentration of fluorophores in tissue of a subject. The techniques disclosed herein are also able to account for variable viewing angles of a fluorophore concentration reference target, non-uniform fluorescence signal detection sensitivity across the field of view of a single image capturing system, and non-uniformities between different image capturing systems. The techniques disclosed herein may help solving the challenging problem of creating a uniform reference standard for fluorescence images of tissue of subjects. By providing a uniform reference standard in physical units, fluorescence signals can be compared across patient groups, and different fluorescence imaging systems.

For example, a method of determining a concentration of a fluorophore in a tissue of a subject, comprises receiving 520 data of a fluorescence image of a fluorophore reference target device, detecting 530 the reference target device and fitting a fluorophore concentration data model based on the data, receiving data of a fluorescence image of a tissue of a subject, and converting 540 the fluorescence image of the tissue of the subject into a fluorophore concentration map indicative of the concentration of the fluorophore in the tissue of the subject, based on the fluorophore concentration data model.

The fluorophore reference target device may be a reference device for calibrating fluorescence imaging systems in fluorescence-guided medical interventions, printed as set forth herein.

The step of receiving 520 the data of the fluorescence image of the fluorophore reference target device and the step of receiving data of a fluorescence image of a tissue of a subject may be performed simultaneously, for example by a single image acquisition with both the fluorophore reference target device and the tissue in the field of view of the imaging system. Alternatively, these steps may be performed sequentially. Preferably both steps are performed under the same or similar conditions. The fluorophore concentration data model may comprise a mapping from detected data values in the fluorescence image of the fluorophore reference target device to corresponding fluorophore concentration values. This mapping may be used in step 540 to convert the fluorescence image of the tissue of the subject into the fluorophore concentration map.

Fig. 5 illustrates aspects of a method 500 of determining the concentration of a fluorophore in a tissue of a subject. The method may comprise a step 510 of placing a fluorophore concentration reference target in the field of view of a fluorescence imaging system. With the fluorophore concentration reference target in the field of view of the fluorescence imaging system, in step 521 , fluorescence images of the fluorophore reference target are captured by the fluorescence imaging system.

In step 520, a computer system receives fluorescence images depicting the fluorophore concentration reference target, the fluorescence images being or having been acquired by the fluorescence imaging system.

In step 530, the computer system detects the fluorophore concentration reference target in one or more of the received fluorescence images, and fits a fluorophore concentration data model to the data. The fluorophore concentration data model maps the pixel values of the areas of the fluorescence images corresponding to the reference patches printed on the fluorophore concentration reference target with different densities, to corresponding fluorophore concentration units. Thus, a model is fitted that maps pixel intensity values of the received fluorescence images to corresponding fluorophore concentration values.

In step 540, the fluorescence images are converted into fluorophore concentration maps by applying the fitted fluorophore concentration data model to the fluorescence images. The fluorophore concentration map indicates, for at least one (tissue) location (pixel) of a fluorescence image, a fluorophore concentration value indicative of a concentration of fluorophore at that location.

These fluorescence images may be generated by the image capturing system in step 541 or 521 , usually at the same time or shortly after or shortly before the image with the fluorophore concentration reference target in it is generated. In step 550, the computer system may extract data attributes that are clinically relevant from the data of the fluorophore concentration map. In step 560, the computer system may control a display to show the extracted data attributes and/or at least part of the fluorophore concentration map.

In some implementations, an example method may be used in step 530 for detecting the region of interest (ROI) representing a fluorophore concentration reference target in the fluorescence images received from a fluorescence imaging system. In some implementations, such a reference target is detected by its shape (e.g., using a contour matching algorithm), color, or a combination thereof. In other implementations, a target may include one or more fiducial markers for automatic detection of the target in image data. In alternative implementations, fiducial markers may separately mark each location of each individual patch printed on the reference target. Alternatively, at least one fiducial marker may mark the location of a plurality of patches as a group.

In some implementations, data markers encoding target metadata may be provided in or on the target. Data fields may be directly encoded in the data marker. Alternatively, a unique identifier may be encoded in or on the reference target, and the field values are stored in a database and retrievable with the unique identifier. A combination of direct encoding and an identifier may also be present. In some implementations, data markers encoding target metadata may be used instead of, or in addition to, fiducial markers for the purpose of locating the target in image data.

Fig. 6 illustrates a part of step 530, namely a method 600 of detecting the reference target and determining the intensity of the fluorescence signal within the detected patches. In some implementations, method 600 may be performed as part of step 530 in Fig 5. Method 600 may be used to determine the intensity of the fluorescence signal within each of the detected patches. The method comprises in step 610, determination of the precise location parameters of the patches. This determination may be performed by detecting the above-mentioned fiducial marker(s) or data markers, or by means of an image detection program. For example, the reference target device may be recognized in the image by image recognition. The locations of the patches relative to the overall location of the reference target device may be stored in a table, so that the patches can be localized within the region of the image showing the reference target device.

The method comprises in step 620, determining the measured fluorescence intensity information for each patch. This step may comprise calculating a value indicative of the pixel intensity at each detected patch location. This value may comprise at least one of an average, median, or peak image intensity of pixels at the detected patch location.

In step 630, an optional check may be performed to validate that the relative fluorescence intensities for each patch meet certain quality criteria 630. If the check succeeds, the determined relative fluorescence intensity information 631 of each patch is output to the next processing step.

In one exemplary embodiment, determining the intensity information of a patch is performed in step 620 by taking the average grayscale pixel value of a square region at the center of the patch. This example process can be expressed by the equation:

wherein I is the intensity of the patch (output in step 631 ), n is the size of the summing region in pixels (a total of (2n+1 )² pixels), and x and y are coordinates of the centroid of the patch.

Quality criteria for the intensity information of a patch may be used in step 630 to reject invalid values and/or to detect bad input images. Such quality criteria may include a fluorescence intensity test, the purpose of which is to filter out patches of which the intensity is too low (e.g., the majority of pixels fall below a pre-defined threshold), oversaturated (e.g., the majority of pixels are above a pre-defined threshold), for which the contrast between the highest and lowest concentrations does not exceed a predefined threshold, or for which the signal-to-noise ratio (SNR) does not exceed a predefined threshold value (e.g., that exhibit too much noise to be measured reliably). If a patch is rejected in step 630, this may have different reasons. One possible reason may be that there is a misalignment of its location parameter and the actual location of the patch in the image data. In such a case, its location parameters may be updated in step 640. Updating a location parameter may include changing the position, or changing the size of the region used to extract the intensity value. After updating the location parameters, the process returns to step 620.

Figure 7 illustrates another part of step 530, namely fitting the fluorophore concentration data model. In some implementations of embodiments disclosed herein, as is illustrated in the example method 700, the method for fitting a data model may comprise retrieving the intensity information, which may be the intensity information output by step 631 , or intensity information obtained in another processing method. Further, the method may comprise, in step 710, obtaining the reference values for fluorophore concentration of each reference patch. These reference values may be set at the time of manufacturing the reference target device, for example. Also the fit parameters to be fitted may be obtained.

In step 720, a data model with the fit parameters is fitted to the data. In step 730, the method optionally comprises validating that the data model meets certain predetermined quality criteria. In step 731 , the method comprises outputting the data model 731 . The output data model may be used in step 240 to convert fluorescence images into fluorophore concentration maps.

The fluorescence intensity information I of each detected patch in the acquired fluorescence image of the fluorophore reference target device may be combined with the known reference concentration values R of the used target. A method of fitting a data model is provided, comprising finding a function M such that

= MI The function M may be computed by performing an appropriate fitting method such as linear regression, weighted linear regression, convex optimization, or the like.

According to an exemplary embodiment, the data model may be described by the equation: logio F(x) m log₁₀ x + fe, wherein F(x) denotes a molar concentration, x is the image intensity, m is the fitted slope and b is a constant. The data model output at step 731 comprises a function F per the above equation and the values of coefficients m and b.

The model described above may work particularly well in case of a sufficient linearity of the fluorescence imaging system and sufficient imaging conditions. In some embodiments, non-linear data models are used in order to overcome any non-linearity of the fluorescence imaging system.

In certain implementations, a goodness of fit score may be determined in step 730 to validate that the data model meets certain predetermined quality criteria. Such a process may comprise generating a goodness of fit score for the data model, comparing it to the quality criteria, and updating the fit parameters in step 740, in order to improve the goodness of fit, or decrease a fit error. Depending on the used data model and fit method, quality criteria for validating the data model, may include setting upper or lower boundaries, as appropriate, for some goodness of fit score such as, for example, a coefficient of determination, a Pearson chi-square test, a mean squared prediction error (MSPE), or the like. Other constraints may be put on the data fit model, such as the constraint that the function F be a monotonic function.

Updating the fit parameters may include omitting at least one of the patches from the input data, and repeating step 720 without the omitted patch. Thus, in some implementations a new data model may be fitted, each with a different patch omitted and the method may further comprise the step of selecting the result with the minimum fit error that passes the quality criteria in step 730. Such an implementation provides robustness to the fitting of a data when input image quality defects, such as a specular reflection, may be present inside of a reference patch.

In some implementations, correction of input data (e.g., fluorescence images) may be performed to compensate for any non-ideal responses of the image capturing system. Data correction may include corrections for spatial non-uniform ity of pixel gain, spatial non-uniform ity of the illumination, non-linear signal response, crosstalk between measurement channels used to image different fluorophores, or a combination thereof. The correction, when applied to the input fluorescence images, may remove non-uniformities, non-linearities or other inaccuracies from the input fluorescence images.

As shown in Fig 5, the method 500 may include step 521 of generating fluorescence images of a fluorophore reference target device, step 520 of receiving the data of the generated images, and step 530 of detecting the reference target device and fitting the data model, at a moment in time before step 541 of generating fluorescence images of tissue of a subject. However, there is no limitation in the order in which the images are generated. Steps 221 and 241 may be performed in any order. The fluorophore reference target device may be placed in the view of the image capturing system in step 210, before capturing its image in step 221. After step 221 the fluorophore reference target device may be optionally removed (not illustrated). Step 530 may be performed before step 540. Step 221 may be performed before step 230. Step 241 may be performed before step 240. However, these steps may be repeated as often as necessary.

In some implementations, generating fluorescence images of a flurorophore reference target device in step 521 , receiving the data fluorescence images of the target device in step 520, and detecting the target device and fitting the data model in step 530 is performed once as a factory calibration activity for a particular imaging device.

In some implementations, generating fluorescence images of a flurorophore reference target device in step 521 , receiving the data fluorescence images of the target device in step 520, and detecting the target device and fitting the data model in step 530 are performed as a periodic field service calibration activity for a particular imaging device.

In some implementations, generating fluorescence images of a flurorophore reference target device in step 521 , receiving the data fluorescence images of the target device in step 520, and detecting the target device and fitting the data model in step 530 are performed at the start, during, or at the end of an imaging session with a particular imaging device.

In some implementations, generating fluorescence images of a fluorophore reference target device in step 521 , receiving the data fluorescence images of the reference target device in step 520, and detecting the reference target device and fitting the data model in step 530 are performed every time the imaging parameters are impactfully changed during an imaging session with a particular imaging device.

In some implementations, generating fluorescence images of a fluorophore reference target device in step 521 , receiving the data fluorescence images of the reference target device in step 520, and detecting the reference target device and fitting the data model in step 530 are performed in real-time for each image of a real-time video stream, or for a significant portion of all images of a real-time video stream (e.g., performing the steps for every 100^th image of a 25 fps video stream results in an update rate of the data model fit of once per four seconds. In any implementation, capturing the fluorescence images with an image capturing system may include placing a reference target device in the field of view of the image capturing device in step 510 at a similar distance from the excitation light projection and image capturing arrangements of the image capturing device as the tissue under investigation.

In some implementations, at least a portion of the method may be performed by a computer system embedded in the imaging system, or located separate from the imaging system. For instance, some or all of the steps 520, 530, 540 and 550 may be performed by a computer. The computer may alternatively be located at an off-site location from the clinical site where the fluorescence imaging system is located. The computer may be alternatively located at a clinical setting but not physically incorporated in an imaging apparatus. It will be understood that, throughout this description, a computer system may be any control unit or computer processor.

Although the invention has been discussed in the foregoing with reference to an exemplary embodiment of the method of the invention, the invention is not restricted to this particular embodiment which can be varied in many ways without departing from the invention. The discussed exemplary embodiment shall therefore not be used to construe the appended claims strictly in accordance therewith. On the contrary the embodiment is merely intended to explain the wording of the appended claims without intent to limit the claims to this exemplary embodiment. The scope of protection of the invention shall therefore be construed in accordance with the appended claims only, wherein a possible ambiguity in the wording of the claims shall be resolved using this exemplary embodiment.

Aspects of the invention are itemized in the following section.

1 . A reference device (1 ) for calibrating a fluorescence imaging system in fluorescence-guided medical interventions wherein the reference device (1 ) comprises at least one reference area (2) comprising at least one fluorescent contrast agent capable of fluorescing when stimulated by electromagnetic waves, characterized in that the at least one fluorescent contrast agent is printed on a at least part of a surface of the reference device (1 ) and configured to exhibit a plurality of fluorescence intensities.

2. The reference device (1 ) according to clause 1 , characterized in that the at least one fluorescent contrast agent comprises a fluorescent dye wherein the fluorescence intensities are invoked by corresponding concentration levels of said fluorescent dye inside the at least one fluorescent contrast agent.

3. The reference device (1 ) according to clause 1 or 2, characterized in that the fluorescent dye is capable of emitting light with a wavelength or wavelengths between 400 nm and 1800 nm when stimulated by electromagnetic waves.

4. The reference device (1 ) according to any one of the preceding clauses, characterized in that the fluorescent dye comprises a near-infrared fluorescent dye capable of emitting light with a wavelength or wavelengths between 600 nm and 950 nm when stimulated by electromagnetic waves.

5. The reference device (1 ) according to any one of the preceding clauses, characterized in that the concentration levels of the fluorescent dye inside the at least one fluorescent contrast agent are between 0.001 nanomolar and 1000 micromolar.

6. The reference device (1 ) according to any one of the preceding clauses, characterized in that the at least one fluorescent contrast agent comprises a solvent-based ink, preferably compatible for use with an inkjet printer or with a padprinter.

7. The reference device (1 ) according to any one of the preceding clauses, characterized in that the at least one fluorescent contrast agent comprises a waxy resin-based ink, preferably compatible for use with a solid ink printer. 8. The reference device (1) according to any one of the preceding clauses, characterized in that the at least one fluorescent contrast agent comprises a granulated ink, preferably compatible for use with a toner printer.

9. The reference device (1 ) according to any one of the preceding clauses, characterized in that the fluorescent dye comprises a derivative of at least one group of cyanines, xanthenes, triarylmethanes, (na)phthalocyanines, (proto)porphyrines, oxazines, (chalcogeno)pyrylium, boron-dipyrromethenes, squaraines, croconaines, polymethines, chromenylium, and flavylium.

10. The reference device (1) according to any one of the preceding clauses, characterized in that the fluorescent dye comprises one of indocyanine green, methylene blue, protoporphyrin IX disodium, fluorescein, Alexa488, BM-104, cy5, cy5.5, cy7, cy7.5, ZW800-1 , asp5354, cypate, DY800, IRdye800CW, IRdye700DX, S0456, ADS680HO and ADS680WS or derivatives thereof.

11. The reference device (1) according to any one of the preceding clauses, characterized in that the fluorescent dye comprises an optically-active nanoparticle, such as a quantum dot, a dye-embedded silica nanoparticles, or gold nanocluster, with a particle size between 1 nm and 1000 nm.

12. The reference device (1) according to any one of the preceding clauses, characterized in that the at least one fluorescent contrast agent comprises a solvent such as alcohol, dimethylformamide, n-methylpyrrolidone or dimethylsulfoxide.

13. The reference device (1) according to any one of the preceding clauses, characterized in that the reference device (1) comprises a substrate comprising at least one of paper, plastic, cellulose, cellulose acetate, PVC, textile or metallic material.

14. The reference device (1) according to any one of the preceding clauses, characterized in that the reference device (1 ) comprises a graduated ruler (3) or scale for measuring a length.

15. The reference device (1) according to any one of the preceding clauses, characterized in that the reference device (1 ) comprises at least one identification label (4) for identifying the fluorescent contrast agent.

16. Use of the reference device of any one of the preceding clauses for providing ground truth fluorescence values when training an artificial neural network. 17. A method for manufacturing a reference device (1 ) for calibrating a fluorescence imaging system in fluorescence-guided medical interventions, characterized in that the method comprises the steps of : providing a substrate (5) comprising at least one of paper, plastic, cellulose, cellulose acetate, PVC, textile or metallic material; and creating at least one reference area (2) on said substrate by: preparing at least one fluorescent contrast agent by dissolving a fluorescent dye in a solvent mixed with a transparent printer-ink, wherein a concentration level of the fluorescent dye inside the at least one fluorescent contrast agent is between 0.001 nanomolar and 10 millimolar; and printing said at least one fluorescent contrast agent on the substrate (5).

18. The method according to clause 17, characterized in that the step of printing the at least one fluorescent contrast agent on the substrate (5) comprises the step of using an inkjet printer or a pad printer or a toner printer, or a solid-ink printer.

19. The method according to clause 18, characterized in that the method comprises the step of providing the fluorescent dye as a near-infrared fluorescent dye.

20. The method according to clause 17-19 characterized in that the method comprises the step of providing the transparent ink as a solvent-based ink.

21 . The method according to any one of clauses 17-20 characterized in that the method comprises the step of providing the fluorescent dye as a derivative of at least one group of cyanines, xanthenes, triarylmethanes, (proto_porphyrines, oxazines, (na)phthalocyanines, boron-dipyrromethenes, squaraines, croconaines, polymethines, chromenylium, (chalcogeno)pyrylium, and flavylium.

22. The method according to any one of clauses 17-21 , characterized in that the method comprises the step of providing the fluorescent dye as one of indocyanine green, methylene blue, protoporphyrin IX disodium, S0456, BM-104, cypate, DY800, ZW800-1 , ASP5354, IRdye800CW, IRdye700DX ADS680HO and ADS680WS.

23. The method according to any one of clauses 17-22, characterized in that the method comprises the step of providing the solvent as one of alcohol, dimethylformamide, n- methylpyrrolidone or dimethylsulfoxide.

24. The method according to clause 17, characterized in that the method comprises the step of providing the fluorescent dye as an optically-active nanoparticle.

25. The method according to any one of clauses 17-24 characterized in that the step of printing the at least one fluorescent contrast agent on the substrate comprises the step of filling a first ink cartridge of said inkjet printer with a first fluorescent contrast agent comprising a first concentration level of a first fluorescent dye and filling at least a second ink cartridge with at least a second fluorescent contrast agent comprising at least a second concentration level of at least a second fluorescent dye.

26. The method according to clause 25, characterized in that the first fluorescent dye and the at least second fluorescent dye are the same.

27. The method according to clause 25 or 26, characterized in that the first concentration level and the at least second concentration level are equal.

28. The method according to any one of clauses 17-27, characterized in that the step of printing the fluorescent contrast agent on the substrate comprises the step of filling at least one ink cartridge exclusively with the transparent ink.

29. The method according to any one of clauses 17-28, characterized in that the method comprises the steps of: creating a computer-readable image by correlating red, green, black (RGB) values of a computer readable image to the cyan, magenta, yellow, key (CMYK) values of an inkjet printer; and using said computer-readable image to print the at least one contrast agent on the substrate.

30. The method according to clause 29, characterized in that the method comprises the step of controlling the fluorescence intensities of the at least one contrast agent by controlling color transparency levels in the computer- readable image.

31. The method according to any one of clauses 17-30, characterized in that the method comprises the step of calibrating the reference device (1 ) by:

- creating a plurality of calibration solution by dissolving the fluorescent dye in a solvent containing between 1 % and 8% weight of albumin, wherein the concentration levels of the fluorescent dye are between 0.001 nanomolar and 1000 micromolar;

- visually comparing the fluorescent intensities in the at least one reference area (2) of reference device (1) to the fluorescent intensities in the plurality of the calibration solution for associating fluorescent intensities in the at least one reference area (2) of calibration device (1 ) to the fluorescent intensities in the plurality of the calibration solution.

32. The method according to any one of clauses 17-31 , characterized in that the method comprises the step of providing a graduated ruler (3) or scale on the reference device (1 ) for measuring a length and/or an image resolution.

33. The method according to any one of clauses 17-32, characterized in that the method comprises the step of providing at least one identification label (4) on the reference device (1 ) for identifying the fluorescent contrast agent.

34. The method according to any one of clauses 17-33, characterized in that the method comprises the step of laminating the substrate (5) using a laminating pouch after printing the at least one fluorescent contrast agent on the substrate.

35. The method according to any one of clauses 17-34, characterized in that the method comprises the step of fixing the at least one fluorescent contrast agent using a fixative agent, such as aldehydes, after printing the at least one fluorescent contrast agent on the substrate.

36. The method according to any one of clauses 17-35, characterized in that the method comprises the step of sterilizing the reference device for medical use.

Claims

1. A method for manufacturing a reference device (1 ) for normalizing a measurement result of a fluorescence imaging system in fluorescence-guided medical interventions, characterized in that the method comprises the steps of : providing a substrate (5); and creating at least one reference area (2) on said substrate by: preparing at least one fluorescent contrast agent by dissolving a fluorescent dye in a solvent mixed with a transparent printer-ink, wherein a concentration level of the fluorescent dye inside the at least one fluorescent contrast agent is between 0.001 nanomolar and 10 millimolar; printing said at least one fluorescent contrast agent on the substrate (5) using a printer, wherein the printing comprises controlling an amount of fluorescent contrast agent that is dispensed from the printer to form a series of discretely separated areas wherein each area has a different fluorescence intensity, or to form a gradient of varying fluorescence intensities of the fluorescent dye.

2. The method of claim 1 , wherein the step of printing the at least one fluorescent contrast agent on the substrate comprises the step of filling a first ink cartridge of said printer with the fluorescent contrast agent.

3. The method according to any one of claims 1 -2 characterized in that the step of printing the at least one fluorescent contrast agent on the substrate comprises the step of filling a first ink cartridge of said printer with a first fluorescent contrast agent comprising a first concentration level of a first fluorescent dye and filling at least a second ink cartridge with at least a second fluorescent contrast agent comprising at least a second concentration level of at least a second fluorescent dye.

4. The method according to claim 3, characterized in that the method comprises printing the first fluorescent contrast agent and the second fluorescent contrast agent on the substrate (5) using the printer.

5. The method according to claim 4, characterized in that the printing comprises controlling an amount of the first fluorescent contrast agent that is dispensed from the printer to form a first series of discretely separated first areas wherein each first area has a different fluorescence intensity of the first fluorescent dye, or to form a first gradient of varying fluorescence intensities of the first fluorescent dye, and controlling an amount of the second fluorescent contrast agent that is dispensed from the printer to form a second series of discretely separated second areas wherein each second area has a different fluorescence intensity of the second fluorescent dye, or to form a second gradient of varying fluorescence intensities of the second fluorescent dye.

6. The method according to any one of claims 3-5, characterized in that the first fluorescent dye and the at least second fluorescent dye are different.

7. The method according to any one of claims 3-6, characterized in that the first concentration level and the at least second concentration level are different.

8. The method according to any one of claims 2-7, characterized in that the step of printing the fluorescent contrast agent on the substrate comprises the step of filling at least one ink cartridge exclusively with the transparent ink.

9. The method according to any preceding claim, characterized in that the fluorescence intensity of the fluorescent dye formed on the substrate corresponds to an amount of the fluorescent contrast agent dispensed from the printer per area of the substrate.

10. The method according to any one of claims 1 -9, characterized in that the method further comprises the step of providing at least one ink cartridge filled with ink having a visible color and controlling the printer to print a visible symbol on at least one region of the substrate.

11 . The method according to any one of claims 3-9, characterized in that the printer is a color printer that can print a color image, and controlling the printer comprises providing control signals associated with a spot color channel of the printer, such as levels of cyan, magenta, yellow, and/or black, to control the fluorescence intensity of each fluorescent dye formed on the substrate.

12. The method according to any one of claims 1 -11 , characterized in that the method comprises the steps of: creating a computer-readable image by correlating red, green, black (RGB) values of a computer readable image to the cyan, magenta, yellow, key (CMYK) values of an inkjet printer; and using said computer-readable image to print the at least one contrast agent on the substrate.

13. The method according to claim 12, characterized in that the method comprises the step of controlling the fluorescence intensities of the at least one contrast agent by controlling color transparency levels in the computer-readable image.

14. The method according to any one of claims 1 -13, characterized in that the method comprises the step of calibrating the reference device (1 ) by:

- creating a plurality of calibration solution by dissolving the fluorescent dye in a solvent, wherein the concentration levels of the fluorescent dye are between 0.001 nanomolar and 1000 micromolar;

- visually comparing the fluorescent intensities in the at least one reference area (2) of reference device (1 ) to the fluorescent intensities in the plurality of the calibration solution for associating fluorescent intensities in the at least one reference area (2) of calibration device (1 ) to the fluorescent intensities in the plurality of the calibration solution.

15. The method according to any one of claims 1 -14, characterized in that the method comprises the step of providing a graduated ruler (3) or scale on the reference device (1 ) for measuring a length and/or an image resolution.

16. The method according to any one of claims 1 -15, characterized in that the method comprises the step of providing at least one identification label (4) on the reference device (1 ) for identifying the fluorescent contrast agent.

17. The method according to any one of claims 1 -16, characterized in that the method comprises the step of laminating the substrate (5) using a laminating pouch after printing the at least one fluorescent contrast agent on the substrate.

18. The method according to any one of claims 1 -17, characterized in that the method comprises the step of fixing the at least one fluorescent contrast agent using a fixative agent, such as aldehydes, after printing the at least one fluorescent contrast agent on the substrate.

19. The method according to any one of claims 1 -18, characterized in that the method comprises the step of sterilizing the reference device for medical use.

20. The method according to any one of claims 1 -19, characterized in that the method comprises cutting the substrate to form a plurality of reference devices after the printing is completed.

21 . The method according to any preceding claim, characterized in that the step of printing the at least one fluorescent contrast agent on the substrate (5) comprises the step of using an inkjet printer or a pad printer or a toner printer, or a solid-ink printer.

22. The method according to any preceding claim, characterized in that the method comprises the step of providing the fluorescent dye as a near-infrared fluorescent dye.

23. The method according to any preceding claim characterized in that the method comprises the step of providing the transparent ink as a solvent-based ink.

24. The method according to any preceding claim characterized in that the method comprises the step of providing the fluorescent dye as a derivative of at least one group of cyanines, xanthenes, triarylmethanes, (proto_porphyrines, oxazines, (na)phthalocyanines, boron-dipyrromethenes, squaraines, croconaines, polymethines, chromenylium, (chalcogeno)pyrylium, and flavylium.

25. The method according to any preceding claim, characterized in that the method comprises the step of providing the fluorescent dye as one of indocyanine green, methylene blue, protoporphyrin IX disodium, S0456, BM-104, cypate, DY800, ZW800-1 , ASP5354, IRdye800CW, IRdye700DX ADS680HO and ADS680WS.

26. The method according to any preceding claim, characterized in that the method comprises the step of providing the solvent as one of alcohol, dimethylformamide, n-methylpyrrolidone or dimethylsulfoxide.

27. The method according to any preceding claim, characterized in that the method comprises the step of providing the fluorescent dye as an optically-active nanoparticle.

28. A reference device (1 ) for calibrating a fluorescence imaging system in fluorescence-guided medical interventions wherein the reference device (1 ) comprises at least one reference area (2) comprising at least one fluorescent contrast agent capable of fluorescing when stimulated by electromagnetic waves, characterized in that the at least one fluorescent contrast agent is printed on at least part of a surface of the reference device (1 ) and configured to exhibit a plurality of fluorescence intensities, wherein an amount of the printed fluorescent contrast agent forms a series of discretely separated areas wherein each area has a different fluorescence intensity, or forms a gradient of varying fluorescence intensities of the fluorescent dye .

29. A method of determining a concentration of a fluorophore in a tissue of a subject, comprising receiving (520) data of a fluorescence image of a fluorophore reference target device printed according to the method of any one of claims 1 -27; detecting (530) the reference target device and fitting a fluorophore concentration data model based on the data; receiving data of a fluorescence image of a tissue of a subject; and converting (540) the fluorescence image of the tissue of the subject into a fluorophore concentration map indicative of the concentration of the fluorophore in the tissue of the subject, based on the fluorophore concentration data model.