JP6834040B2 - Quantification of absolute blood flow in tissues using fluorescence-mediated photoelectric volumetric pulse wave recording - Google Patents

Description

The present invention generally relates to the field of optical evaluation of blood flow in tissue using photoelectric volumetric pulse wave recording (PPG), and in particular, quantification of blood flow in tissue including microvascular blood flow in tissue. Regarding evaluation.

Perfusion refers to the flow of blood in and out of the capillary bed of tissue. Quantification of tissue perfusion is important for clinicians across many surgical and non-surgical disciplines. A simple binary assessment (flow vs. non-flow) may be appropriate for some clinical applications, but quantification of perfusion in standard measurements is desirable in many other clinical applications. To date, a quantitative assessment of tissue perfusion remains unknown.

Photoelectric volumetric pulse wave recording (PPG) is an optical technique that can be used to estimate changes in blood volume in microvessels, and PPG-based techniques include pulse rate, oxygen saturation, blood pressure, and cardiac output. It has been introduced into commercially available medical equipment for evaluation. A typical output of such a device is the PPG waveform corresponding to the heartbeat of the subject. Despite the relatively widespread application of PPG technology to such medical devices, PPG has not been used to provide measurements in standardized units when assessing blood flow. PPG technology with such potential will allow certain measurements of blood volume in tissue, including perfusion measurements, to be made in standardized units of volume / unit time / tissue area. This is of great value to clinicians, and such measurements will allow direct site-to-site and inter-subject comparisons.

According to the first aspect of the present invention, there is provided a method for measuring a change in blood volume in a tissue with time. The method is to excite a fluorescent agent in the blood, such as indocyanine green (ICG), and to obtain a light intensity signal that changes over time between the beating flow of blood through the tissue. The pulsatile flow has diastole and systole, similar to conventional photoelectric pulse wave recording, to obtain and to obtain measurements of changes in blood volume in tissues over time. Includes processing the acquired light intensity signal that changes over time.

In the first embodiment, the method may eliminate the step of administering the fluorescent agent to the subject.

Further, in the first embodiment, the method may exclude correlating the measurement of changes in blood volume in tissue over time with physiological, diagnostic, or pathological parameters.

According to the second embodiment, an apparatus for measuring a change in blood volume in a tissue with time is provided. This device is a means for exciting a fluorescent agent in blood, for example, indocyanine green (ICG), and a means for acquiring a light intensity signal that changes with time between the beating flow of blood passing through a tissue. The pulsatile flow has diastole and systole, similar to conventional photoelectric plethysmography, acquired by means and to obtain measurements of changes in blood volume in tissue over time. Includes means for processing a time-varying light intensity signal.

According to the third form, a kit for measuring the time course of blood volume in the tissue is provided, and the kit includes the device of the second form and a fluorescent agent such as ICG.

According to the fourth embodiment, a fluorescent agent is provided for use in a method for measuring a change in blood volume in a target tissue over time, the method of which is according to the first embodiment.

In various forms of the invention, the modified Beer-Lambert's law applies to diastole and systole of the pulsatile flow of blood through the tissue.
ΔL = ln [(IeΦ-Im) / (IeΦ-Ip)] (εC) ^-1
ΔL is the change in the total thickness of the blood layer in a given tissue, Ie is the intensity of the excitation light that excites the fluorescent agent in the blood, Φ is the quantum efficiency of the fluorescent agent, and Im is the tissue. The intensity of the light intensity signal that changes over time during the minimum expansion period of the beating flow of blood passing through, Ip is the intensity of the light intensity signal that changes over time during the maximum systole of the beating flow of blood passing through the tissue, ε is , Molar absorptiometry of the fluorescent agent, C gives the instantaneous molar concentration of the fluorescent agent in blood.

In various embodiments of the present invention, the instantaneous molar concentration of the fluorescent agent in blood is preferably determined by utilizing concentration-mediated changes in the fluorescent emission spectrum of the fluorescent agent. Concentration-mediated changes in the fluorescent emission spectrum of optical brighteners include monotonous spectral shifts.

In various embodiments of the present invention, utilizing concentration-mediated changes in the fluorescence emission spectrum of a fluorescent agent preferably comprises selecting first and second spectral bands in the fluorescence emission spectrum of the fluorescent agent. And to obtain the first and second intensity of the fluorescence emission integrated at the wavelength for each of the second spectral bands, to calculate the ratio of the first and second intensity, and to obtain the value of C from the ratio. To derive and include. In various embodiments, the first spectral band includes wavelengths in the range of about 780 nm to about 835 nm, or a portion thereof, and the second spectral band is from about 835 nm to about 1000 nm, or a portion thereof. Includes wavelengths in the range.

According to one embodiment, selecting the first and second spectral bands causes one of the first and second intensities to change monotonically with C and one of the first and second intensities with C. It does not change. According to other embodiments, selecting the first and second spectral bands has a different rate, although the first and second intensities increase monotonically with C. According to still another embodiment, selecting the first and second spectral bands causes various embodiments in which the first intensity increases monotonically with C and the second intensity decreases monotonically with C. In, the instantaneous molar concentration of the fluorescent agent in the blood ranges from about 2 μM to about 10 mM.

Any feature of the invention described above may be applied in any combination to any form of the invention, unless otherwise required in the context.

Further optional features of the present invention will be described below.

The accompanying drawings show embodiments of the present invention.
FIG. 1 is a diagram schematically showing the use of conventional photoelectric plethysmography (PPG) in which a fingertip sensor is used to measure pulse rate, blood oxygen saturation, or both. FIG. 2 is a diagram showing a fluorescence emission spectrum of an indocyanine green (ICG) dye that shifts to a longer wavelength as the molar concentration of the dye in blood increases according to an embodiment. FIG. 3 shows an embodiment in which the instantaneous molar concentration of the fluorescent agent in blood is determined by utilizing the spectral shift of the fluorescence emission spectrum of the fluorescent agent, and one of the first and second intensities becomes monotonous with the concentration. It is a figure which shows that the 1st and 2nd spectral bands are selected so that one of the 1st and 2nd intensities changes and does not change with density. FIG. 4 shows an embodiment in which the instantaneous molar concentration of the fluorescent agent in blood is determined by utilizing the spectral shift of the fluorescence emission spectrum of the fluorescent agent, although the first and second intensities increase monotonically with the concentration. It is a figure which shows that the 1st and 2nd spectral bands are selected so that they have different speeds. FIG. 5 shows an embodiment in which the instantaneous molar concentration of the fluorescent agent in blood is determined by utilizing the spectral shift of the fluorescence emission spectrum of the fluorescent agent, the first intensity increasing monotonically with the concentration and the second. It is a figure which shows that the 1st and 2nd spectral bands are selected so that an intensity decreases monotonically with a density | concentration. FIG. 6 is a diagram showing an exemplary device for measuring a change in blood volume in a tissue with time according to an embodiment. FIG. 7 is a diagram showing an exemplary lighting module according to an embodiment. FIG. 8 is a diagram showing an exemplary fluorescence radiation acquisition module according to an embodiment. FIG. 9 shows a first spectral band in the range of about 820 to about 840 nm (“SWL” indicates short wavelength) and a second spectral band in the range of about 840 nm to about 900 nm (“LWL” indicates long wavelength). It is a figure which shows the exemplary relationship between the ratio of the ICG fluorescence intensity of a spectral band, and the instantaneous molar concentration of ICG. FIG. 10 is a diagram showing another embodiment of the excitation means for fluorescent excitation of the fluorescent agent of the apparatus in FIG.

Here, implementations and embodiments of various embodiments and modifications of the present invention will be referred to in detail, examples of which are shown in the accompanying drawings.

Conventional photoelectric volumetric pulse wave recording (PPG) can estimate changes in tissue blood volume by detecting changes in the amount of red or near-infrared light passing through the tissue. As the blood volume in the tissue expands and contracts during the cardiac pressure pulse corresponding to the subject's heartbeat, the amount of light absorbed by the blood volume increases and decreases, respectively. As shown in FIG. 1, for example, the total blood volume in the fingertip blood vessels is the smallest during the cardiac pressure pulse diastole and the largest during systole. Although it may be used to measure pulse rate and blood oxygen levels, the application of this PPG technique is not configured to make volume measurements in standardized units.

In order to be able to provide volume-measured microvascular blood flow measurements in standardized units, the PPG waveform metric must be associated with changes in blood volume in the tissue in a known and repeatable manner. .. It is possible to establish a deterministic relationship for the application of the modified Beer-Lambert's law (also known as the Beer-Lambert-Lambert-Lambert method). Beer-Lambert's law correlates the attenuation of light passing through a medium with the length of the path through the medium and its absorption rate, a relationship used in conventional PPGs. Traditional PPGs are performed by passing light of near-infrared wavelength through tissue (eg, fingertips), but the need for tissue transillumination makes the application of this method more general for volumetric blood flow measurements in tissues. In some cases, it is practically restricted. According to certain embodiments, the present invention utilizes modified Beer-Lambert's law to enable such blood flow measurements using fluorescence wavelengths radiated by fluorescent agents such as fluorescent dyes. Such dyes are preferentially bound to plasma, for example, thereby allowing both the light beam source and the fluorescence detector to be placed on the surface of the tissue. For example, the fluorescence emitted from the pigment-labeled plasma component of blood conforms to the modified Veil-Lambert's law, and by solving the equation for optical path length and quantifying each parameter, the fluorescence-mediated PPG is microscopic without transillumination. Volumetric blood flow measurements, including vascular blood flow measurements, can be provided.

Thus, in contrast to conventional PPG techniques, the present invention measures standardized units (eg, volume) that measure changes in blood volume within a tissue over time and present these changes as blood flow, including microvessels. (/ Unit time) provides a fluorescence-mediated photoelectric volumetric pulse wave recording method (FM-PPG). In FM-PPG, according to various embodiments, the detected fluorescence intensity is proportional to the instantaneous concentration of the fluorescent agent in the blood (eg, the fluorescent agent in plasma) and thus microvascular blood flow or perfusion. It can be used to determine blood flow in tissues, including. Blood flow within a tissue is generally understood as an increase in the total amount of blood flowing into an anatomical structure or region; blood flow includes tissue perfusion or microvascular blood flow and is of an anatomical structure or region. The amount of blood flowing through the capillaries of the blood vessel bed. In various embodiments, the methods and devices of the present invention are used to measure blood flow in tissue, and more specifically to measure perfusion or microvascular blood flow in tissue. In various embodiments, the use of the methods and devices of the present invention includes the ability to distinguish between blood flow and microvascular blood flow.

According to one embodiment of the present invention, there is provided a method for measuring a change in blood volume in a tissue over time. The method is to obtain a time-varying light intensity signal, including a time-varying fluorescence intensity signal, between exciting a fluorescent agent in the blood and the pulsatile flow of blood through the tissue. The pulsatile flow has a diastole and a systole, which is similar to the conventional photoelectric pulse wave recording method, and includes acquisition. The method applies the modified Beer-Lambert's law to diastole and systole to process the acquired time-varying light intensity signal in order to obtain a measurement of blood volume in tissue over time. Further prepare.

In various embodiments, a suitable fluorescent agent is capable of circulating blood and circulates with a component of the blood, such as plasma in the blood, which fluoresces when exposed to the appropriate excitation light energy. Drugs that can be used). In addition, fluorescent agents exhibit concentration-mediated changes in their fluorescence emission spectrum. In various embodiments, the concentration-mediated changes include a monotonous spectral shift in the fluorescent emission spectrum of the fluorescent agent. An example of a fluorescent agent is a fluorescent dye, which includes any non-toxic fluorescent dye whose concentration exhibits a monotonous spectral shift. In certain embodiments, the fluorescent dye is a dye that emits light in the near infrared spectrum. In certain embodiments, the fluorescent dye is, for example, a tricarbocyanine dye such as indocyanine green (ICG). In other embodiments, the fluorescent dye further uses fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalaldehyde, fluorescamine, rosebengal, tripanblue, using the appropriate excitation wavelength for each dye. It may be fluorogold or a combination thereof. In some embodiments, analogs or derivatives of fluorescent dyes may be used. For example, fluorescent dye analogs or derivatives include fluorescent dyes that are chemically modified but retain the ability to fluoresce when exposed to light energy of the appropriate wavelength.

One form of measuring the change in blood volume in a subject's tissue over time is to administer the fluorescent agent to the subject so that the fluorescent agent circulates with the blood in the tissue so that the blood flows through the tissue. To be equipped. In various embodiments, the fluorescent agent may be administered intravenously to the subject at an appropriate concentration for contrast, eg, as a bolus administration. In various embodiments, the fluorescent agent may be injected into the vein, artery, microvascular system (eg, capillary bed) or a combination thereof of interest so that it circulates in the microvascular system. In embodiments where multiple fluorescent agents are used, such agents may be administered simultaneously, eg, in a single bolus, or sequentially, eg, in a separate bolus. In some embodiments, the fluorescent agent may be administered by catheter. In certain embodiments, the fluorescent agent may be administered to the subject less than an hour before making measurements according to the various embodiments. For example, the fluorescent agent may be administered to the subject less than 30 minutes before making the measurement. In yet another embodiment, the fluorescent agent may be administered at least 30 seconds before the measurement. In yet other embodiments, the fluorescent agent may be administered at the same time as performing the measurements described in relation to the various embodiments.

In another embodiment, the method may eliminate any step of administering the fluorescent agent to the subject.

Fluorescent agents may be provided as lyophilized powders, lyophilized solids or liquids. In certain embodiments, the fluorescent agent can be provided in vials (eg, sterile vials) that can be reconstituted to the appropriate concentration by administering the sterile solution with a sterile syringe. Reconstitution may be carried out with any suitable carrier or diluent. For example, the fluorescent agent may be reconstituted with water immediately prior to administration. In various embodiments, any diluent or carrier that keeps the fluorescent agent in solution may be used. As an example, in certain embodiments where the fluorescent agent is ICG, the fluorescent agent may be reconstituted with water. In some embodiments, once the fluorescent agent has been reconstituted, it may be mixed with additional diluents and carriers. In some embodiments, the fluorescent agent may be combined with another molecule, such as a protein, peptide, amino acid, synthetic polymer or sugar, for example to enhance solubility, stability, contrast properties or a combination thereof. Additional buffers, including Tris, HCl, NaOH, phosphate buffer, HEPES, may optionally be added.

In various embodiments, the fluorescent agent may be administered at various concentrations to achieve the desired circulating concentration in the blood. For example, if the fluorescent agent is ICG, it may be administered at a concentration of about 2.5 mg / mL, achieving a circulating concentration of about 5 μM to about 10 μM in the blood. In various embodiments, the upper limit concentration of the administration of the fluorescent agent is the concentration at which the fluorescent agent becomes clinically toxic in the circulating blood, and the lower limit concentration is the device detection limit for detecting the fluorescent agent in the circulating blood. .. In various other embodiments, the upper limit concentration of fluorescent agent administration is the concentration at which the fluorescent agent self-quenches. In a further embodiment, the lower concentration limit for fluorescent agent administration is a concentration that makes it difficult to detect the fluorescent agent with conventional contrast techniques. For example, when the fluorescent agent is ICG, the circulating concentration of the fluorescent agent may be in the range of 2 μM to about 10 mM.

The method of measuring the time course of blood volume in a tissue further comprises acquiring a time-varying light intensity signal during the pulsatile flow of blood through the tissue. In various embodiments, the pulsatile flow results from a cardiac pressure pulse generated by a heartbeat or simulated heartbeat (eg, the use of a blood pump). Pulsatile flow includes diastole and systole. Furthermore, the diastole and systole are similar to conventional photoelectric plethysmography.

The method further comprises processing the acquired time-varying light intensity signal (eg, time-varying fluorescence intensity signal) to provide a measurement of the time course of blood volume in the tissue, modified Beer-Lambert. The law applies to diastole and systole. The modified Beer-Lambert's law for radiated fluorescence is described as:
ΔL = ln [(IeΦ-Im) / (IeΦ-Ip)] (εC) ^-1
ΔL is the change in the total thickness of the blood layer in a given tissue, Ie is the intensity of the excitation light that excites the fluorescent agent, Φ is the quantum efficiency of the fluorescent agent, and Im is the blood passing through the tissue. The intensity of the light intensity signal that changes with time during the minimum period of expansion of the beating flow, Ip is the intensity of the light intensity signal that changes with time during the maximum systole of blood passing through the tissue, and ε is the fluorescent agent. Molar absorption coefficient, C, is the instantaneous molar concentration of the fluorescent agent in blood.

As shown in FIG. 2, the emission spectrum of the ICG dye in whole blood is different for different molar concentrations of the dye. In various embodiments, the instantaneous molarity of the fluorescent agent is determined by utilizing concentration-mediated changes in the fluorescent emission spectrum of the fluorescent agent. Concentration-mediated changes include a monotonous spectral shift in the fluorescent emission spectrum of the fluorescent agent.

In various embodiments, utilizing concentration-mediated changes in the fluorescence emission spectrum of a fluorescent agent involves selecting the first and second spectral bands of the fluorescence emission spectrum of the fluorescent agent (eg, as shown in FIG. 3). ), Obtaining the first and second intensities of fluorescence emission integrated at wavelengths for each of the first and second spectral bands, and calculating the ratio of the first and second intensities, and calculation. It is provided with deriving the value of C in the modified Vale-Lambert's law from the given ratio.

In various embodiments, the first and second spectral bands may be selected in several ways. According to one embodiment, in the first and second spectral bands, one of the first and second intensities changes monotonically (increases or decreases) with C, and one of the first and second intensities is C. Selected so that it does not change with. For example, as shown in FIG. 3, the intensity of fluorescent radiation integrated at wavelengths for any band selected in range B remains unchanged as the concentration of fluorescent agent increases. In addition, the intensity of the fluorescence emission integrated at the wavelength for any band selected in range A decreases with C. Therefore, the A / B bandwidth ratio decreases with C.

According to another embodiment, the first and second spectral bands are chosen to have different velocities, although the first and second intensities diminish monotonically with C. For example, as shown in FIG. 4, the intensity of the fluorescence emission integrated at the wavelength for any band selected in range B decreases with C, but is integrated at the wavelength for any band selected in range A. The intensity of fluorescence emission decreases more slowly with C. Therefore, the A / B bandwidth ratio decreases with C.

According to yet another embodiment, the first and second spectral bands are selected such that the first intensity monotonically increases with C and the second intensity monotonically decreases with C. For example, as shown in FIG. 5, the intensity of the fluorescence emission integrated at the wavelength for any band selected in region B increases with C, but is integrated at the wavelength for any band selected in range A. The intensity of fluorescence emission decreases with C. Therefore, the A / B bandwidth strength ratio decreases with C, but decreases at a greater rate than in previous embodiments.

In various embodiments, when the fluorescent agent is, for example, ICG, the first spectral band comprises a wavelength in the range of about 780 nm to about 835 nm, or a portion thereof, and the second spectral band is, for example, about 835 nm. Includes wavelengths in the range of about 1000 nm, or a portion thereof.

By selecting the first and second spectral bands as described in relation to the various embodiments, it is clinically identifiable over a range of clinically expected concentrations of fluorescent agent in circulating blood. A change in ratio can be achieved and the instantaneous molar concentration C of the fluorescent agent can be determined.

In various embodiments, the method further comprises correlating the measurement of changes in blood volume in a tissue with time to biological parameters, physiological parameters, diagnostic parameters, pathological parameters, or a combination thereof. You can. In another embodiment, the method derives from the measurement of changes in blood volume in tissues over time a measurement of changes in biological, physiological, diagnostic, pathological, or combinations thereof. Including that. In various embodiments, examples of biological, physiological, diagnostic, pathological, or combinations thereof are indicators or conditions of tissue, conditions of interest, or combinations thereof (eg, atherosclerosis). Includes arteriosclerosis, oxygen supply, cardiac output).

In various other embodiments, the method may eliminate correlating measurements of changes in blood volume in tissues over time with physiological, diagnostic, or pathological parameters.

According to another embodiment of the present invention, there is provided an apparatus for measuring a change in blood volume in a tissue with time. This device is a means for exciting a fluorescent agent in blood and a means for acquiring a light intensity signal that changes with time between a beat flow of blood passing through a tissue (the beat flow is, for example, a heartbeat or a blood pump). The pulsatile flow has diastole and systole, similar to conventional photoelectric pulse wave recording methods, such as means and tissues. A means for processing the acquired time-varying light intensity signal is provided in order to obtain a measurement of the blood volume in the body over time. Applying the modified Beer-Lambert's law to diastole and systole:
ΔL = ln [(IeΦ-Im) / (IeΦ-Ip)] (εC) ^-1
Obtain as described in connection with the embodiment of the method.

In various embodiments of the device, the instantaneous molar concentration C of the fluorescent agent is determined by the use of concentration-mediated changes, including monotonous spectral shifts in the fluorescent emission spectrum of the fluorescent agent. In various embodiments, the present application is the selection of the first and second spectral bands of the fluorescent emission spectrum of the fluorescent agent and the first and first of the fluorescent emissions integrated at wavelengths for the first and second spectral bands. It includes the acquisition of the intensity of 2 and the calculation of the ratio to the first and second intensities and the derivation of the value of C from that ratio.

According to one embodiment, the selection of the first and second spectral bands is such that one of the first and second intensities changes monotonically with C and one of the first and second intensities does not change with C. Is. According to another embodiment, the first and second intensities monotonically increase at a rate different from C. According to yet another embodiment, the first intensity monotonically increases with C and the second intensity monotonically decreases with C. Examples of these embodiments are shown in FIGS. 3 to 5. In various embodiments, the first spectral band includes wavelengths in the range of about 780 nm to about 835 nm, or a portion thereof, and the second spectral band includes wavelengths in the range of about 835 nm to about 1000 nm, or a portion thereof. Including.

With reference to FIG. 6, exemplary embodiments of the device 10 for measuring changes in blood volume in tissues over time are schematically shown. The device 10 includes an excitation means 12 that excites a fluorescent agent 14 in blood in the tissue, an acquisition means 16 that acquires a light intensity signal that changes with time during the pulsating flow of blood passing through the tissue, and an in-tissue The processing means 18 for processing the time-varying light intensity signal acquired to provide the measurement of the time-varying blood volume is provided.

In various embodiments, the excitation means 12 comprises, for example, a lighting module that includes a fluorescence excitation source and is configured to generate excitation light having an intensity suitable for exciting the fluorescent agent 14 and a wavelength suitable for excitation. .. FIG. 7 shows an exemplary lighting module according to an embodiment. The illumination module 20 includes a laser diode 22 (eg, including one or more fiber-coupled diode lasers) to provide excitation light for exciting the fluorescent agent 14 (not shown). Examples of other sources of excitation light that can be used in various embodiments are one or more LEDs, arc lamps, or other light sources of sufficient intensity and suitable wavelength to excite the fluorescent agent 14 in the blood. Including technology. For example, the excitation of the fluorescent agent 14 in blood, where the fluorescent agent 14 is a fluorescent dye having near-infrared excitation and radiation properties, was conducted by DILAS Diode Laser (Germany) at one or more 793 nm and conducted. , A single bar, fiber-coupled laser diode module may be used.

In various embodiments, the light output from the source of the excitation light is projected through an optical element (ie, one or more optics) to the output used to illuminate the tissue area of interest. Mold and guide. The molded optics may consist of one or more lenses, optical guides, and / or diffractive elements to ensure a flat field over the entire field of view of the fluorescence emission acquisition module. In certain embodiments, the fluorescence excitation source is selected to emit light at a wavelength close to the absorption maximum of the fluorescent agent 14 (eg, a fluorescent dye such as ICG). For example, referring to the embodiment of the illumination module 20 of FIG. 7, the output 24 from the laser diode 22 passes through one or more focusing lenses 26 and is then generally available, for example, from Newport Corporation (USA). It passes through a homogenized optical pipe 28 such as an optical pipe. Finally, the light passes through an optical diffraction element 32 (ie, one or more optical diffusers), such as a ground glass diffraction element, also available from Newport Corporation (USA), for example. Power to the laser diode 22 itself is provided by a high current laser driver, such as that available from Lumina Power, Inc. (USA). The laser may be operated in pulse mode during the image acquisition process. In this embodiment, an optical sensor such as a solid photodiode 30 is incorporated into the illumination module 20 to sample the illumination intensity produced by the illumination module 20 via scattered or diffuse reflections from various optical elements. In various embodiments, additional sources of illumination may be used to provide guidance when aligning and positioning the module over a region of interest.

Returning to FIG. 6, in various embodiments, the acquisition means 16 includes, for example, a fluorescence emission acquisition module for acquiring a fluorescence signal (eg, a light intensity signal that changes over time) from the fluorescent agent 14. The fluorescence emission acquisition module includes an image sensor. Referring to FIG. 8, an exemplary embodiment of the fluorescence radiation acquisition module 40 for acquiring a fluorescence signal such as a light intensity signal that changes over time from the fluorescent agent 14 (not shown) is shown. As shown in FIG. 8, the fluorescent radiation 42 from the fluorescent agent 14 (not shown) in the blood is collected and a 2D solid-state image sensor (eg, an image) using imaging optics 48a, 48b and 48c. Focus on the sensor 44 and the image sensor 46). The solid-state image sensor may be a charge-coupled device (CCD), CMOS sensor, CID or similar 2D sensor technology. An optical filter 50 (which may include multiple optical filters of various configurations) is used to remove residual and reflected excitation light to ensure that only fluorescence radiation is recorded on image sensors 44 and 46. To do. In this embodiment, the dichroic optical filter 52 is used to divide the fluorescence emission spectrum of the fluorescent agent 14 into two spectral channels (eg, first and second spectral bands). In this embodiment, the dichroic optical filter 52 divides the total fluorescence emission substantially equally between the two spectral channels, the short wavelength channel collects light with a wavelength below the maximum fluorescence emission, and the long wavelength channel has the maximum fluorescence emission. Designed to collect the above light. The charges generated from the optical signals converted by the image sensors 44 and 46 are converted into an electrical video signal, including both digital and analog video signals, by appropriate readout and amplification electronics in the fluorescence emission acquisition module 40.

In the embodiment shown in FIG. 8, only two image sensors 44 and 46 are used, but a preferred choice of two spectral bands may utilize the signals converted by the two sensors in two beneficial ways. .. Fluorescent radiation over wavelengths in one band increases monotonically with the fluorescent agent concentration, and integrated fluorescent radiation at wavelengths in another band decreases monotonically with the fluorescent agent concentration, as shown in FIG. First, the signals from the two image sensors 44 and 46 may be combined to obtain total fluorescence image signal intensity. This makes it possible to generate the highest quality (lowest noise) fluorescence image. Second, the image signals from these two spectral bands are proportional to each pixel and can determine the instantaneous molar concentration of the fluorescent agent 14 in the blood. Molarity is an essential parameter in determining changes in blood volume in tissues over time. The images from the two image sensors 44 and 46 show the same field of view for each pixel. Further, when the range of variation of the ratio as shown in FIG. 9 is expanded, the determination of the instantaneous concentration of the fluorescent agent 14 is the result by utilizing the selection of the spectral band as described in relation to the various embodiments. Will be more accurate as.

Returning to FIG. 6, in various embodiments, the processing means 18 was calculated by, for example, analyzing a time-varying light intensity signal and performing a plethysmographic calculation of the time-dependent change in blood volume in the tissue. Includes a processor module (not shown) that outputs information to the appropriate display and / or recording device, or a combination thereof. In various embodiments, the processor module includes any computer or computing means, such as, for example, a tablet, laptop, desktop, network computer, or dedicated stand-alone microprocessor. The inputs are obtained from, for example, the image sensors 44, 46 of the radiation acquisition module 40 shown in FIG. 8, the solid photodiode 30 in the lighting module 20 of FIG. 7, and any external control hardware such as a footswitch or remote control. The output is supplied to the laser diode driver and the optical alignment assist device. In various embodiments, the processor module may have the ability to store the image sequence in an internal memory such as a hard disk or flash memory, allowing post-processing of the acquired data. In various embodiments, the processor module may have an internal clock to allow control of various elements and ensure accurate timing of illumination and sensor shutters. In various other embodiments, the processor module may provide a graphic representation of user inputs and outputs.

In various other embodiments, the apparatus 10 shown in FIG. 6 may instead include excitation means 12A for fluorescence excitation of the fluorescent agent 14 (not shown), as shown in FIG. The excitation means 12A includes a first excitation source 90 and a second excitation source 92 for providing excitation light for exciting the fluorescent agent 14 (not shown). The output from each excitation source passes through beam shaping and smoothing optics, as described in connection with the embodiments described above. In this embodiment, the acquisition means (not shown) is not only an optical filter (not shown) for eliminating residual and reflected excitation light, but also fluorescence similar to that described in connection with the previous embodiment. It also includes a fluorescence emission acquisition module consisting of acquisition and imaging optics. The optical system preferably focuses the collected fluorescence on a single solid-state image sensor, which is read out by the processing module at each frame.

With reference to the embodiments of FIGS. 6-8, in the process, the region of interest is located under both the excitation means 12 including the illumination module 20 and the acquisition means 16 including the fluorescence radiation acquisition module 40 of the apparatus 10. The object is then positioned so that the illumination module 20 creates a substantially uniform illumination field over the area of interest. In various embodiments, images of the region of interest may be acquired for the purpose of background deduction prior to administration of the fluorescent agent 14 to the subject. For example, to do this, the operator presses a remote switch or foot control, or via a keyboard on the processing module (not shown) of processing means 18 of device 10 in FIG. May start. As a result, the excitation source (eg, the laser diode 22 of the illumination module 20 in FIG. 7) is turned on and the shutter sequence for the image sensor (eg, the image sensors 44, 46 of the fluorescence emission acquisition module 40 in FIG. 8) is To be started. When operating in the pulse mode of the present embodiment, each image sensor is read out at the same time as the laser pulse. In this way, the maximum fluorescence emission intensity is recorded and the signal-to-noise ratio is optimized. In this embodiment, the fluorescent agent 14 is administered to the subject and delivered to the region of interest via arterial flow. Image acquisition is started, for example, immediately after administration of fluorescent agent 14, and an image of fluorescence returning from the entire region of interest is acquired during the entry of fluorescent agent 14. The fluorescence emission from the region of interest is collected by the image optical system in front of the fluorescence emission acquisition module 40. The residual and reflected excitation light is attenuated by an optical filter (eg, the optical filter 50 of FIG. 8).

In the embodiment of FIG. 8, a dichroic optical filter 52 is used to divide the acquired total fluorescence into two selected spectral channels, as described in relation to the various embodiments. In a single exposure, the images recorded by the sensors 44 and 46 are read out and sent to the processor module (not shown) of the processing means 18 of device 10 shown in FIG. In various embodiments, the processor module may average across adjacent pixels within each frame and across multiple consecutive frames before performing the perfusion calculation. Images recorded on each of the two spectral channels are compared and the ratio of fluorescence intensity of each channel is calculated across the kernel of the field of view. The kernel may be a single pixel or an array of pixels in the field of view. The concentration of ICG in the kernel is calculated based on the calculated ratio and the previous calibration of the device. The combined signals from both image sensors 44 and 46 are then used, along with the measurement of the light illumination intensity measured by the sampled solid photodiode 32 in the illumination module 20 of FIG. 7, to calculate the total fluorescence intensity and vary. The amount of blood in the kernel is determined through the application of modified Vale Lambert's law, as described in connection with the above embodiments. This process is repeated over the entire field of view, and the resulting perfusion (blood flow) measurement is displayed to the user on demand, for example as a grayscale or pseudo-color image, or for later analysis. Saved for.

In yet another embodiment, a kit for measuring changes in blood volume in tissue over time is provided, the kit being described above in connection with various embodiments and described above in connection with various embodiments. Includes fluorescent agents such as ICG.

In yet another form, a fluorescent agent is provided for use in a method for measuring changes in blood volume in a subject's tissue over time, and various forms of the method are described above.

Although the present invention has been illustrated and described in connection with various embodiments illustrated and described in detail, it is not intended to be limited to the details shown, and thus various modifications and structural changes are made to the present invention. Can be done in any way without departing from the range of. Various variations of the described embodiments with respect to form, components, steps, arrangement of details, and procedures of operation deviate from the scope of the present invention in any way, as in other embodiments of the present invention. It may be done without, and will be apparent to those skilled in the art with reference to this description. Therefore, the appended claims are intended to extend to such modifications and embodiments so as to fall within the true scope of the invention. With respect to the terms "eg" and "such" and their grammatical equivalents, the phrase "and without limitation" is understood as is unless explicitly stated otherwise. As used herein, the singular forms "a", "an" and "the" include a plurality of referents unless expressly indicated otherwise in the context.

Claims (35)

A method of operating a device for measuring a change in the amount of blood in a tissue of a target tissue with time, and the method of operation is
Capturing a sequence of fluorescent images of the tissue from the excited fluorescent agent in the blood in the blood vessels that feed the tissue of the tissue of interest.
Acquiring a fluorescence intensity signal that changes over time during the pulsatile flow of blood passing through the tissue from the sequence of fluorescence images of the tissue.
The pulsatile flow has a diastole and a systole.
The acquired fluorescence intensity signal includes a first fluorescence intensity emitted by the fluorescent agent in the blood in the tissue during diastole.
The acquired fluorescence intensity signal includes a second fluorescence intensity emitted by the fluorescent agent in the blood in the tissue during systole.
The first and second fluorescence intensities are associated with the measurement of blood volume during the diastole and systole, respectively.
By utilizing the concentration-mediated change in the fluorescence emission spectrum of the fluorescent agent, the molar concentration of the fluorescent agent in the blood can be calculated.
The change in the total thickness of the blood layer is calculated based on the first and second fluorescence intensities and the determined molar concentration, which is the tissue hemoperfusion in the tissue. It is a quantitative measurement of
To generate a modified image of the tissue of the subject, at least in part, based on the quantitative measurement, the modified image of the tissue of the subject is a fluorescent image of the tissue. To display the same area as the above sequence,
Displaying the modified image and assisting in the clinical evaluation of the tissue of the subject.
Operation method including. The operation method according to claim 1, wherein the concentration-mediated change comprises a spectrum shift of the fluorescence emission spectrum of the fluorescent agent. The calculation of the molar concentration of the fluorescent agent in the blood
Selecting the first and second spectral bands in the fluorescence emission spectrum of the fluorescent agent
To obtain the first and second intensities of fluorescence radiation integrated at wavelengths for each of the first and second spectral bands.
Calculating the ratio of the first and second intensities and
To derive the value of the molar concentration of the fluorescent agent in the blood from the ratio,
The operation method according to claim 1, further comprising. In the first and second spectral bands, one of the first and second intensities changes monotonically with the molar concentration, and one of the first and second intensities changes with the molar concentration. The operation method according to claim 3, which is selected not to be used. The operation method according to claim 3, wherein the first and second spectral bands are selected so that the first and second intensities increase monotonically with the molar concentration but at different velocities. 3. The first and second spectral bands are selected such that the first intensity monotonically increases with the molar concentration and the second intensity monotonically decreases with the molar concentration, claim 3. How it works. The operation method according to claim 3, wherein the first spectral band includes a wavelength in the range of 780 nm to 835 nm, and the second spectral band includes a wavelength in the range of 835 nm to 1000 nm. Obtaining the first and second intensities of fluorescent radiation,
Recording the fluorescence emission in the first spectral band with an image sensor,
Recording the fluorescence emission in the second spectral band with an image sensor,
3. The operation method according to claim 3. Combining the data from the recording of the fluorescence radiation in the first spectral band and the data from the recording of the fluorescent radiation in the second spectral band into an image of the tissue including the visual field of the tissue. The operation method according to claim 8, further comprising. The operation method according to claim 1, wherein the fluorescent agent is indocyanine green (ICG). The operation method according to claim 10, wherein the molar concentration is in the range of 2 μM to 10 mM. The operation method according to claim 1, further comprising correlating the measurement of the time course of the amount of blood in the tissue with physiological parameters, diagnostic parameters, pathological parameters, or a combination thereof. Further comprising repeating the calculation of changes in the total blood layer thickness of the same region of the tissue.
The operation method according to claim 1, wherein generating the modified image of the tissue of interest is at least partially based on repeating the calculation of the same region of the tissue. The operation method according to claim 1, wherein the modified image is either a grayscale image or a pseudo color image. A device for measuring a change in the amount of blood in a tissue of a target tissue over time.
A light source configured to excite the fluorescent agent in the blood in the blood vessels supplied to the tissue of the tissue of interest.
A sensor configured to capture a sequence of fluorescent images of the tissue and obtain a fluorescence intensity signal that changes over time from the sequence of angiographic images of the tissue during the beating flow of blood through the tissue. And
The pulsatile flow has a diastole and a systole.
The acquired fluorescence intensity signal includes a first fluorescence intensity emitted by the fluorescent agent in the blood in the tissue during diastole.
The acquired fluorescence intensity signal includes a second fluorescence intensity emitted by the fluorescent agent in the blood in the tissue during systole.
The first and second fluorescence intensities are associated with the measurement of blood volume during the diastole and systole, respectively, with the sensor.
It ’s a processor,
By utilizing the concentration-mediated change in the fluorescence emission spectrum of the fluorescent agent, the molar concentration of the fluorescent agent in the blood can be calculated.
The change in the total thickness of the blood layer is calculated based on the first and second fluorescence intensities and the determined molar concentration, which is the tissue hemoperfusion in the tissue. It is a quantitative measurement of
To generate a modified image of the tissue of the subject, at least in part, based on the quantitative measurement, the modified image of the tissue of the subject is a fluorescent image of the tissue. To display the same area as the above sequence,
To display the modified image and to assist in the clinical evaluation of the tissue of the subject.
With a processor configured to do
Including equipment. 15. The apparatus of claim 15, wherein the concentration-mediated change comprises a spectral shift of the fluorescence emission spectrum of the fluorescent agent. The calculation of the molar concentration of the fluorescent agent in the blood
Selecting the first and second spectral bands in the fluorescence emission spectrum of the fluorescent agent
To select the first and second intensities of fluorescence radiation integrated at wavelengths for each of the first and second spectral bands.
Calculating the ratio of the first and second intensities and
Derivation of the molar concentration value from the ratio and
15. The device of claim 15. In the selection of the first and second spectral bands, one of the first and second intensities changes monotonically with the molar concentration, and one of the first and second intensities is the molar concentration. The device of claim 17, which is made to remain unchanged with. 17. The apparatus of claim 17, wherein the selection of the first and second spectral bands is such that the first and second intensities increase monotonically with the molar concentration, but at different rates. 17 The selection of the first and second spectral bands is such that the first intensity monotonically increases with the molar concentration and the second intensity monotonically decreases with the molar concentration. The device described. The apparatus according to claim 17, wherein the first spectral band includes wavelengths in the range of 780 nm to 835 nm, and the second spectral band includes wavelengths in the range of 835 nm to 1000 nm. The device according to claim 15, wherein the fluorescent agent is indocyanine green (ICG). 22. The apparatus of claim 22, wherein the molar concentration is in the range of 2 μM to 10 mM. A claim, wherein the light source includes a lighting module that includes a fluorescence excitation source, the fluorescence excitation source being configured to generate excitation light having a suitable intensity and a suitable wavelength for exciting the fluorescent agent. 15. The apparatus according to 15. 24. The apparatus of claim 24, wherein the fluorescent excitation source includes a first excitation source and a second excitation source. The illumination module further includes an optical element configured to form and guide the excitation light exiting the illumination module and provide a uniform field of excitation light over the entire region of interest including said tissue of interest. 24. The apparatus according to claim 24. 26. The apparatus of claim 26, wherein the optical element comprises a lens, an optical guide, a diffraction element, or a combination thereof. The device according to claim 15, wherein the sensor includes a fluorescence radiation acquisition module including an image sensor. The fluorescence radiation acquisition module is arranged in front of the image sensor configured to capture, filter, and direct the time-varying light intensity signal generated by the fluorescent agent to the image sensor. 28. The apparatus of claim 28, further comprising an optical element. The device according to claim 15, wherein the processor includes a processor module. 30. The processor module is configured to control the operation of the light source, to control the operation of the sensor, or to control a combination thereof. apparatus. The sensor configured to acquire a fluorescence intensity signal that changes over time during the beating flow of blood through the tissue includes an image sensor to acquire the first and second intensity of fluorescence emission. Is
Recording the fluorescence emission in the first spectral band with an image sensor,
Recording the fluorescence emission in the second spectral band with an image sensor,
17. The device of claim 17. The processor captures data from the fluorescence emission recording in the first spectral band and data from the fluorescence emission recording in the second spectral band into an image of the tissue including the visual field of the tissue. 32. The apparatus of claim 32, further configured to synthesize. The processor is further configured to repeat the calculation of changes in the total blood layer thickness of the same region of the tissue.
15. The apparatus of claim 15, wherein producing the modified image of the tissue of interest is at least partially based on repeating the calculations in the same region of the tissue. The device according to claim 15, wherein the modified image is either a grayscale image or a pseudo color image.