Mapping and quantification of tissue oxygenation levels following aminolevulinic acid administration using delayed fluorescence imaging

Abstract

A system for imaging oxygen concentration in tissue or diffusion media using a protoporphyrin IX fluorescent probe includes a pulsed excitation light that excites a fluorophore and a gated electronic camera synchronized to the pulsed light. The gated electronic camera captures an immediate fluorescence image in an immediate fluorescence time window overlapping with a light pulse from a pulsed light source and a delayed fluorescence image after the light pulse. An image processor receives the immediate and delayed fluorescence images and generates therefrom an image indicative of tissue oxygenation. In another embodiment, a method for generating an oxygen level image of tissue includes acquiring immediate and delayed fluorescence images of tissue using a pulsed excitation light illuminator and a gated camera. The method includes determining a ratio image from the delayed fluorescence image and the immediate fluorescence image, the ratio image providing an oxygen level image of the tissue.

Description

Government Rights Clause

[0001] This invention was made with Government support under Grant No. P01CA084203 awarded by the National Institutes of Health. The Government has certain rights in the invention. Claims of Priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 405,839, filed September 12, 2022. This application also claims priority to U.S. Provisional Patent Application No. 63 / 402,011, filed August 29, 2022, which claims priority to U.S. Provisional Patent Application No. 63 / 320,072, filed March 15, 2022. The entire contents of the foregoing provisional patent applications are incorporated herein by reference. [Background technology]

[0003] Surgical oncology utilizes fluorescence as a contrast mechanism, and there are already several commercial systems and injectable tracers approved for human use. Most of the tracers used simply indicate vascular perfusion, since the injected dyes are designed for intravenous injection and are designed to indicate areas of high vascular capillary perfusion or areas of capillary leakage.

[0004] Advances are being made in fluorescent imaging agents that characterize tissue metabolism, each with its own advantages and limitations. Protoporphyrin IX (PPIX) is a fluorescent molecule produced in almost all human and other mammalian tissues through the heme synthesis pathway, an endogenous or native molecule stimulated by administration of its precursor, aminolevulinic acid (ALA). ALA can be administered locally to tissues or intravenously or orally to patients. In all three cases, metabolic absorption occurs over the course of several hours to generate PPIX in cells throughout the body.

[0005] PPIX is produced, for example, by the rapid metabolism of ALA in glioma tumor tissue, and is produced faster in glioma tissue than in surrounding normal brain tissue, allowing for contrast between glioma and normal brain tissue by rapid fluorescence imaging. Similarly, it is absorbed by skin and cavity tissues and incorporated into the production of PPIX, making it commonly prescribed as a photodynamic therapy agent for skin lesions and used to visualize bladder cancer lesions during cystoscopy.

[0006] Therefore, immediate fluorescence imaging with protoporphyrin IX fluorophore in human and other mammalian tissues following ALA administration has been used to distinguish tumor tissue from the surrounding normal tissue. It is approved for use during cystoscopy and brain glioma surgery to ensure adequate surgical margins. When tissue is illuminated with blue light, the immediate bright pink fluorescence of PPIX can be visually confirmed.

[0007] Unfortunately, strong PPIX production is also observed in many glandular and other active tissues, limiting the PPIX concentration contrast between many tumor tissues and surrounding normal tissues. Fluorescence imaging of these lesions with standard systems imaging standard immediate or prompt fluorescence does not provide sufficient contrast for some applications. Thus, PPIX is of little value as a fluorescence imaging guide in complex surgeries, with only a limited number of lesions preferentially enhancing fluorescence. Furthermore, although PPIX is produced in many tissues, it is eventually delivered to the bloodstream, where it is redistributed throughout the body within hours and spreads via the bloodstream to most organs, resulting in reduced long-term effective contrast.

[0008] When illuminated by excitation light, most fluorophores exhibit a fluorescent response with immediate or prompt fluorescence, but a small number of fluorophores exhibit a type of fluorescence emission called delayed fluorescence. This delayed fluorescence is due to the energy structure of the molecule, where electronic energy crosses into a triplet state and then back to the singlet state. This energy transfer delays the release of fluorescent energy, and the lifetime of the triplet state determines the duration of the emission. The triplet state of some molecules is quenched by oxygen in the environment, so the lifetime and emission intensity are directly affected by the oxygen level in the environment near the molecule. Marek Scholz, Jason R. Gunn, Geoffrey P. Luke, Brian W. Pogue et al., "Imaging singlet oxygen feedback delayed fluorescence and lysosomal permeability in in vivo tumors during photodynamic therapy with aluminum phthalocyanine," J. Biomed. Opt. 25(6), 063806 (2020), doi: 10.1117 / 1.JBO.25.6.063806., note that the delayed fluorescence emission of aluminum phthalocyanine differs between tissues with high and low concentrations of singlet oxygen.

[0009] Many tumor tissues grow rapidly and have limited capillary penetration. As a result, many tumor tissues contain hypoxic regions, where tissue hypoxia is defined as tissue with oxygen levels lower than those required for optimal cell growth. These tissues usually secrete growth factors that stimulate blood vessel growth, and these growth factors can be targeted by chemotherapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0010] In one embodiment, aminolevulinic acid is administered to a mammal several hours before surgery.Tissue of the mammal is observed under immediate and delayed PPIX fluorescence, and the ratio of delayed fluorescence to immediate fluorescence in a pixel is mapped as a ratio image.This ratio image is displayed to indicate the location of hypoxic tissue in the tissue.In an embodiment, this system is used to help identify tumors in tissue and to assist in surgical removal of tumors.

[0011] In one embodiment, a system for imaging oxygen concentration in tissue includes a pulsed light source operable at an excitation wavelength of a protoporphyrin IX (PPIX) fluorophore in the tissue and configured to illuminate the tissue with a light pulse; a gated electronic camera synchronized with the pulsed light source and configured to capture a delayed fluorescence image of the tissue in at least one delayed fluorescence time window beginning after the end of the light pulse from the pulsed light source and not overlapping with the light pulse from the pulsed light source; and an image processor coupled to receive the instantaneous and delayed fluorescence images from the gated electronic camera, the image processor configured to generate an image indicative of oxygenation of the tissue. The gated electronic camera is configured to capture an instantaneous fluorescence image in an instantaneous fluorescence time window that overlaps with the time window of the light pulse from the pulsed light source. A filter device is disposed between the tissue and the electronic camera sensor, the filter device configured to block light at the excitation wavelength of PPIX while capturing the fluorescence image, while passing the fluorescent emission of PPIX to the gated electronic camera.

[0012] In another embodiment, a method for generating an oxygen level image of tissue includes using an illuminator and a gated camera configured to provide pulsed fluorescence excitation light and capturing an instantaneous fluorescence image of the tissue and a delayed fluorescence image of the tissue, where the instantaneous fluorescence image is acquired during the pulse of fluorescence excitation light and the delayed fluorescence image is acquired a non-overlapping pulse after the pulse of fluorescence excitation light. The method also includes determining a ratio image from the delayed fluorescence image and the instantaneous fluorescence image, where the ratio image provides an oxygen level image of the tissue. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1A is a block diagram of a system for recognition of hypoxic tissues by delayed fluorescence imaging.

[0014] [Figure 1B] FIG. 1B is a block diagram of an alternative system for recognition of hypoxic tissue by delayed response fluorescence imaging.

[0015] [Figure 1C] FIG. 1C is a block diagram of an alternative camera system that may be used in the systems shown in either FIG. 1A or FIG. 1B.

[0016] [Diagram 2] FIG. 2 is a block diagram of a fast-gated image intensified electronic camera that can be used in the system of FIG. 1A or FIG. 1B, or as the delayed fluorescence camera of FIG. 1C.

[0017] [Diagram 3] FIG. 3 is a timing diagram showing the camera gates (or shutter windows) associated with the capture of immediate and delayed fluorescence images.

[0018] [Figure 4] FIG. 4 is a flow chart showing the operation of the system.

[0019] [Diagram 5] Figure 5 shows the ratio of delayed to immediate fluorescence plotted against time when anesthetized nude mice bearing human pancreatic tumors were exposed to a first pulse of low carbon dioxide concentration and a second pulse of higher carbon dioxide concentration.

[0020] [Figure 6] FIG. 6 shows that the ratio of delayed to immediate fluorescence recovers rapidly after palpation of normal tissue, whereas recovery is significantly slower in tumor tissue.

[0021] [Figure 7] FIG. 7 shows the recovery rates of human pancreatic tumors and surrounding normal tissues on the backs of nude mice. [Figure 8A] FIG. 8A shows the recovery rates of human pancreatic tumors and surrounding normal tissues on the backs of nude mice. [Figure 8B] FIG. 8B shows the recovery rates of human pancreatic tumors and surrounding normal tissues on the backs of nude mice. [Figure 8C] FIG. 8C shows the recovery rates of human pancreatic tumors and surrounding normal tissues on the backs of nude mice. [Figure 8D] FIG. 8D shows the recovery rates of human pancreatic tumors and surrounding normal tissues on the backs of nude mice.

[0022] [Figure 9] FIG. 9 shows an image of another anesthetized mouse with an overlay of a delayed / early fluorescence ratio image of a human pancreatic tumor arising in that mouse.

[0023] [Figure 10] FIG. 10 shows a comparison of contrast between tumor tissue and normal tissue in terms of immediate fluorescence, delayed fluorescence, and delayed fluorescence / immediate fluorescence ratio. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] ALA is a drug approved in several formulations for topical or oral use, which is metabolized in tissues to protoporphyrin IX (PPIX), which then fluoresces strongly in skin and glioma lesions. However, strong contrast is not obtained in typical tumor surgery, as many glandular or metabolically active tissue areas adjacent to the tumor show strong PPIX fluorescence along with the tumor, and the contrast between tumor and normal tissue is poor, as tumor and normal tissue have similar PPIX concentrations. However, this molecule also emits a rather weak but detectable delayed fluorescence, which is greatly amplified in hypoxic areas.

[0025] Therefore, the delayed fluorescence signal is an indicator of tissue oxygenation, especially in areas of low tissue oxygenation or hypoxic tissue. While the prompt fluorescence and image contrast of the immediate fluorescence are low, the delayed fluorescence image contrast is often strong in areas of tissue that are inherently hypoxic or where temporary hypoxia has been induced by gentle compression or palpation.

[0026] Human and other mammalian tissues are diffusion media. In diffusion media other than mammalian tissue, PPIX, or other oxygen-sensitive fluorophores, may be added for the purpose of sensing or mapping oxygen concentrations.

[0027] In a surgical microscope embodiment, a system 100 (FIG. 1A) for imaging and quantifying tissue oxygenation through delayed fluorescence provides an illuminator 102 that illuminates tissue, or other diffusing media, with pulses of light at a fluorescence excitation wavelength suitable for stimulating fluorescence emission from protoporphyrin IX (PPIX) and delivers light to a surgical field 104. In some embodiments, the illuminator 102 includes a pulsed laser operable at a fluorescence emission wavelength. During imaging of tissue oxygenation, the illuminator 102 does not illuminate the tissue with light at the PPIX fluorescence emission wavelength. Light from the surgical field 104, including fluorescence emission from a tumor 108 in the tissue 110, passes through an imaging lens 106 into a body 112 of the surgical imaging system, where a diverter 114 deflects the light through a filter device 116 to a high-speed, high-sensitivity gated camera 118. The high-speed, high-sensitivity gated camera 118 provides electronic images to an image capture unit 120 coupled to an image processor 122 having a memory 124. The pulse sequencer 126 controls the timing of the high speed, high sensitivity gated camera 118 and the pulsed emission by the illuminator 102, thereby synchronizing the on-time of the gated camera with the light pulse from the illuminator 102 and allowing the timing relationship between the light pulse and the on-time of the gated camera to be set.

[0028] In some embodiments, the filter device 116 is a filter changer, while in other embodiments the filter device is a tunable optical bandpass filter that can be set to pass light at the fluorescence emission wavelengths of fluorophores of interest, such as PPIX or indocyanine green (ICG), and block light at the fluorescence excitation wavelengths. In many embodiments, the high speed, high sensitivity gated camera 118 is a high speed gated image intensifier camera formed from a gated image intensifier 140 and an electronic camera 142. In some embodiments, the filter device 116 is a fixed optical bandpass filter that can be set to pass light at the fluorescence emission wavelengths and block ambient light as well as light at the fluorescence excitation wavelengths of fluorophores of interest, such as PPIX or ICG.

[0029] An alternative laparoscopic embodiment 150 (FIG. 1B), such as an endoscope, laparoscope, or similar embodiment that does not require direct viewing of the surgical field through the eyepieces of the device, has an illumination device 152 coupled to an efferent optical fiber or fiber bundle 154 configured to provide light 156 to the tissue 110 and, if present, the tumor 108. As with the embodiment of system 100, in a fluorescence or delayed fluorescence / hypoxia imaging mode, the illuminator 152, 102 provides light at least one fluorescence excitation wavelength, to the exclusion of a fluorescence emission wavelength; in other modes, such as a structured light imaging mode that may be used to determine optical parameters of the tissue 110, light of other wavelengths may be provided by the illuminator 152, 102.

[0030] In endoscopic, laparoscopic and some other embodiments, light 158 ​​from the operative field or tissue in a body cavity or passageway viewed through the endoscope or laparoscope passes through a lens 160 into a coherent fiber bundle 162 and then through another lens 164 and a filter device 166 into a high speed, high sensitivity camera 167. In these embodiments, the diverter 114 is not required since direct viewing through the eyepiece is not supported. The high speed, gated, high sensitivity camera 167 may be an image intensified gated camera, in embodiments, including a high speed, gated image intensifier 168 and an electronic camera 170. These embodiments 150 have electronics similar to the embodiment of FIG. 1A, with the camera 167 providing an electronic image to an image capture unit 120 coupled to an image processor 122 having memory 124. A pulse sequencer 126 controls the timing of the high speed, high sensitivity gated camera 167 and the emission of light by the illuminator 152. The endoscopic and laparoscopic embodiments 150 may be particularly used during minimally invasive surgery in addition to diagnostic and screening procedures.

[0031] In an embodiment, the illuminator 102 is a pulsed laser and the high speed, high sensitivity gated camera 118 is a combination of a high speed, gated image intensifier 250 (FIG. 2) and an electronic camera 252. In an alternative embodiment, the high speed, high sensitivity gated camera 118 incorporates a high speed, single photon avalanche photodiode camera. In yet another alternative embodiment, the high speed, high sensitivity gated camera 118 incorporates a high sensitivity complementary metal oxide semiconductor (CMOS) camera, and in one particular alternative embodiment, the high speed, high sensitivity gated camera 118 is a multi-pulse integrating high sensitivity CMOS camera.

[0032] In some embodiments, the filter device 116, 166 is a filter changer that includes a PPIX emission filter having a passband that blocks light from the illuminator 102, 152 at a fluorescence excitation wavelength suitable for use with PPIX and allows PPIX fluorescence emission to pass. In some embodiments, the filter changer includes an indocyanine green (ICG) emission filter that passes light at an ICG emission wavelength but blocks light from a second illuminator that emits light at a wavelength suitable for stimulating indocyanine green fluorescence emission but does not emit light at an ICG emission wavelength. In some embodiments, the filter changer includes a neutral density filter that allows imaging under PPIX or ICG excitation wavelength illumination. In some embodiments, the filter device 116 is integrated inside the camera through a filter on the camera sensor that receives light only at the emission band of PPIX, or on an individual photosensor of the sensor.

[0033] In alternative embodiments, the filter device 116, 166 is a tunable filter with a passband that is electronically settable not only for the PPIX excitation wavelength, but also for the PPIX emission wavelength. In some alternative embodiments, the passband of the tunable filter can also be set for the ICG emission and excitation wavelengths. This allows the tunable filter to remain in place while the system is performing immediate and delayed PPIX fluorescence imaging, or in some embodiments, ICG fluorescence imaging, and performing reflected or structured light imaging at both the PPIX and ICG excitation wavelengths to determine the optical properties of the tissue. When the tunable filter is used with white light provided by the illuminators 102, 105 operating in different modes, or additional illuminators (not shown), the system can provide full hyperspectral reflectance imaging.

[0034] In some embodiments, a third illuminator (not shown) provides white light illumination so that the cameras 118, 167 can obtain images in normal light, and in embodiments such as system 100 (FIG. 1A), the diverter 114 can be altered to pass light through the eyepiece 132, allowing the surgeon to view the surgical field.

[0035] In an alternative embodiment, the cameras 118, 167 and the filter arrangement 166 may be replaced by multiple cameras with fixed filters 190 and dichroic mirrors 192, as shown in FIG. 1C. In the embodiment of FIG. 1C, the dichroic mirror 192 deflects the light at the fluorescence emission wavelengths to a high-speed, high-sensitivity electronic camera 194, which may be of any of the types described herein with reference to the camera 118 of FIG. 1A or the camera 167 of FIG. 1B. The light at the fluorescence excitation wavelengths is deflected by a second dichroic mirror 198 to a second camera 196 dedicated to imaging the light at the fluorescence excitation wavelengths. The light at other wavelengths passes through the neutral density filter of the filter 190 to a third electronic camera 199, which may be a standard color camera configured to prepare a background image. In this embodiment, the dichroic mirrors 192, 198 and the filter 190 (if present) function as the filter arrangements 116, 166.

[0036] Once images of immediate fluorescence from the tissue, delayed fluorescence from the tissue, tissue under excitation wavelength light, or tissue under white light are captured by the image capture unit 120, they are processed by an image processor 122 operating under the control of code 134 in memory 124 to form a processed image 138 which is displayed to the surgeon on a display 139.

[0037] In some embodiments, the illuminator 102 provides uniform illumination throughout the surgical field and is configured to do so during immediate and delayed fluorescence imaging.

[0038] In an alternative embodiment, known herein as a structured light embodiment, the illuminator 102 is adapted to provide a "structured light" illumination sequence of PPIX fluorescence excitation and fluorescence emission wavelengths, in addition to providing relatively uniform PPIX fluorescence excitation across the entire surgical field, with at least two light patterns of different spatial frequencies, each incorporating alternating light and dark bars of at least three phase offsets. Images are captured by the image capture unit 120 at the excitation wavelength and under instantaneous fluorescence conditions, and processed by the image processor 122 under control of code 134 in memory 124 to provide a three-dimensional model of the optical parameters of the tissue 110 at both the excitation and fluorescence wavelengths. The optical properties extracted from the images include the absorption and scattering coefficients of the tissue at both the fluorescence emission and fluorescence excitation wavelengths, and these optical properties are stored in the memory 124. Methods for extracting these optical parameters from images obtained with structured light have been disclosed in our prior papers and patent applications, where it has been proposed to estimate the depth of fluorescent inclusions, such as tumors 108, within the tissue 110 being observed by the system. These optical properties may be used by the image processor 122 to correct the immediate and delayed fluorescence emission images for absorption and scattering in the tissue and provide an image of the estimated depth and amount of the fluorophores PPIX and / or ICG in the tissue. In these embodiments, in addition to generating and displaying 416 a flat two-dimensional ratio image of the hypoxic tissue, after generating a three-dimensional model of the tissue's optical parameters, the system observes the delayed fluorescence images under at least three phases of the fluorescent stimulation light structured at at least two spatial frequencies and processes the images to generate a three-dimensional map of the ratio of delayed and immediate fluorescence emission throughout the tissue 110, thereby providing a three-dimensional map of the hypoxic tissue highlighting the tumor 108.

[0039] In the hypoxic tissue identification method 400, after ALA is administered and an incubation period for PPIX production is allowed, or if native tissue PPIX is relied upon, optionally mechanical pressure is applied to the tissue to be imaged (401), which is then removed. The filter device 116, 166 is set to pass PPIX fluorescent light while blocking PPIX excitation wavelength light (402). The pulsed excitation wavelength illuminator 102, 152 and fast gated high sensitivity camera 167, 118 operate under the control of the pulse sequencer 126 in a first mode for capturing instantaneous fluorescence, in which the illuminator 102, 152 is turned on (404) to provide one or more pulses 302 (FIG. 2) of PPIX excitation wavelength light.

[0040] In certain, but not all, embodiments, the pulse is 20 microseconds in duration and has a frequency of 500 Hertz, providing 500 microwatts of illumination per square centimeter of tissue 110. During the pulse 302, the camera 118 is gated on (304) and captures (406) an instantaneous fluorescence image before the illuminator 102, 152 is turned off (408) at the end of the pulse. In certain embodiments, the image intensifier of the camera 118, 167 is gated on for approximately 100 nanoseconds at approximately the midpoint of the 20 microsecond illumination pulse 302. The pulsed excitation wavelength illuminator 102, 152 and fast gated high sensitivity camera 118, 167 operate in a second mode to repeat additional pulses 302 of excitation light, with the camera 118 held off (electronic shutter closed) during the pulses 302, held off through a predetermined delay interval 306 (410), and then gated on 308 (electronic shutter open) for an image capture window that does not overlap with the pulse 302 of PPIX excitation light or any subsequent pulses of PPIX excitation light to capture a delayed fluorescence image (412). In a particular embodiment, the image capture window for the delayed fluorescence image is 1.975 milliseconds in length. In one embodiment, the delay interval 306 is 2 microseconds, but in alternative embodiments is adjustable up to 50 microseconds, with a corresponding reduction in the image capture window for the delayed fluorescence image to prevent overlap of the capture of the delayed fluorescence image with the subsequent excitation light pulse. Thus, an immediate fluorescence image is captured during the pulse 302 of excitation light, and a delayed fluorescence image is captured during a valid image capture window that ends after the pulse 302 of excitation light ends and before the leading edge of the next pulse of excitation light. In certain embodiments, the delayed fluorescence images are summed over 50 repetitions of pulse and image capture to provide an average delayed fluorescence image that is used in further processing. In other embodiments, summation or integration of the delayed fluorescence is performed over different numbers of cycles of excitation light on, excitation light off, delay, and opening of the electronic shutter window of the camera to read the delayed fluorescence from the tissue. In yet another embodiment, a similar integration is used when reading the immediate fluorescence images.The summation or integration may be digital if a single photon avalanche photodiode (SPAD) detector is used in the gated camera 118, or analog if a high sensitivity multi-pulse integrating CMOS image sensor is used in the gated camera 118. In some embodiments using a high sensitivity multi-pulse integrating CMOS image sensor or an intensified camera with a fast pulse image intensifier in front of the CMOS camera, the image sensor may be read out after multiple sets of excitation light and the camera electronic shutter open window. In one embodiment, once the immediate and average delayed fluorescence images are available, the image processor 122 is configured to enhance them by subtracting a background image and performing median filtering, and then calculate the ratio (414) of the intensity of each pixel of the enhanced delayed fluorescence image to the enhanced immediate fluorescence image at the same pixel. This ratio is then denoised to form a ratio image (416) that is displayed. Both the delayed fluorescence image and the ratio image show the oxygenation of the tissue. In one embodiment, a ratio is calculated for pairs of immediate and delayed fluorescence images captured adjacent in time, after which additional pairs of immediate and delayed fluorescence images are captured and ratioed to allow observation of dynamic changes in tissue oxygenation. In an alternative embodiment, the imaging sequence 402-416 is repeated to provide an oxygen wash-in sequence after removal of mechanical pressure, where oxygen wash-in may be reduced in areas with poor blood flow, such as certain tumor tissues.

[0041] In a particular embodiment using pancreatic tumors grown in mice, mechanical pressure 401 was applied by palpation and it was observed that delayed fluorescence was enhanced in the tissue for up to 5 minutes after pressure was removed, thus enhancing the delayed fluorescence images and the image of the ratio of delayed to immediate fluorescence.

[0042] In some embodiments, the turn-off time at the trailing edge of the excitation light pulse is less than 50 nanoseconds. In some embodiments, a programmable delay is used between the turn-off of the excitation light pulse and the opening of the electronic shutter of the high-speed gated camera. This delay can be programmed from 1 microsecond to 50 microseconds, and in a particular embodiment is set to 2 microseconds.

[0043] In embodiments in which the fast gated high sensitivity camera 167,118 is an intensified gated camera, the gating of the image intensifier 168,140 is performed as part of gating the camera to respond to light within a particular electronic shutter window.

[0044] In one embodiment, the predetermined delay interval 306 is 2 microseconds to allow the instantaneous fluorescence to decay. In alternative embodiments, the delay interval may be between 1 microsecond and 100 microseconds. In one embodiment, a grayscale image obtained using a filter device 116, 166 set to pass excitation wavelength light is colored according to the ratio image to provide a false color image highlighting hypoxic tissue for display (418).

[0045] In one embodiment, the system of Figures 1-4 is used to determine cancer tissue during surgical treatment of a subject. Most cancerous regions are known to have small, sometimes microscopic areas of hypoxic tissue interwoven throughout the cancerous tissue, resulting from extensive cell proliferation and limited capillary growth and perfusion. Indeed, some chemotherapy regimens act by inhibiting the growth of new capillaries into such tissue. The system described herein is operated to provide images of these hypoxic tissue regions to image the hypoxic nodules of cancer and to guide the surgeon's efforts to remove these nodules with adequate surgical margins.

[0046] In an embodiment, the delayed fluorescence camera gate 308 is turned on for several hundred microseconds after the excitation light pulse is completed. In an embodiment, the light is integrated over several intervals after the excitation light pulse. This drug and device combination provides the ability to view hypoxic tissue in vivo and is utilized to guide interventions such as surgical resections and other procedures.

[0047] In alternative embodiments where the camera is time gated, there is no filter device 116, 166. In these embodiments, the excitation light is completely off during the camera on-time while performing delayed and immediate fluorescence imaging, and delayed vs. immediate fluorescence ratio imaging is not supported.

[0048] We believe that displaying the ratio of delayed to immediate fluorescence will be less sensitive to changes in PPIX concentration in the tissue and to changes in tissue illumination than displaying only delayed fluorescence images. Both delayed and ratio images are representative of tissue oxygenation. This approach is not easily achieved with current systems that simply image immediate fluorescence intensity during the excitation light exposure time.

[0049] In imaging of some tissues and cancerous tumor types,108 PPIX observed as both immediate and delayed fluorescence is endogenous to the tissue, whereas in other tissues and cancer types, PPIX observed as both immediate and delayed fluorescence is expected to be amplified in the tissue by administration of ALA approximately 3–6 h prior to the start of surgery.

[0050] In some embodiments, a second fluorophore, such as ICG, is administered as a bolus while illuminator 130 provides light at an excitation wavelength suitable for ICG to provide and display wash-in and / or wash-out image sequences that can provide the surgeon with an indication of tissue perfusion (420).

[0051] In certain embodiments, the illuminator 102 is configured to emit an excitation wavelength light pulse less than 1 millisecond in length, and in some embodiments 20 microseconds in length. In certain embodiments, the gated camera "shutter" window can be moved in time relative to the excitation wavelength light pulse by a variable delay having a time resolution of less than 100, and in one embodiment, 1 microsecond.

[0052] In some embodiments, the delayed fluorescence image is displayed instead of or before calculating the ratio image, allowing for a simpler image display.

[0053] Use of the above system is premised on imaging tissues where ALA has been administered to produce some degree of PPIX within the tissue, however, in some tissues, the native PPIX signal within the tissue itself produces sufficient delayed fluorescence for imaging.

[0054] Although the above-mentioned system is designed to image hypoxic tissues through delayed fluorescence, in some cases, hypoxia can be induced or increased in tumor tissues by compressing or palpating the tumor tissue or the tissues surrounding it. This palpation induces a temporary inhibition of capillary blood flow, and it is known that tumor tissues take longer to recover from a hypoxic state to a normal oxygen supply state than normal healthy tissues. Thus, there exists a transient hypoxic state that can be imaged with delayed fluorescence that is amplified by applying pressure to the tissue.

[0055] Although this system is designed to image delayed fluorescence from PPIX, in some cases early after ALA administration, insufficient PPIX is produced within the tumor. In such cases, redistribution of PPIX through the blood may bring PPIX into the tumor through passive blood transport and diffusion to the region. Thus, in tumors that do not natively produce sufficient PPIX by metabolizing ALA, imaging of delayed fluorescence can be improved by waiting longer after ALA administration.

[0056] In a particular embodiment, the filter device 116, 166 is set to a passband of 697±37 nanometers during PPIX immediate and delayed fluorescent imaging.

[0057] In some embodiments, images representative of tissue oxygenation are used to guide surgical treatment of tissue in a subject or to diagnose a medical condition in a subject. In particular, since low oxygen levels in tissue are often indicative of malignant tissue, images representative of tissue oxygenation may be used to guide the surgical removal of tissue having low oxygen levels.

[0058] To assist the surgeon in locating tissues with low oxygenation levels relative to other tissues in the field of view, in an embodiment, a reflectance image of the tissue is obtained by setting the filter device 116, 166 to pass light at a fluorescent excitation wavelength and capturing a reflectance image during a pulse of fluorescent excitation light from the illuminator 102, 152 during a brief period when immediate and delayed fluorescent imaging is not occurring. In an alternative embodiment, the filter device 116, 166 is set to a neutral density filter and the illuminator 102, 152 is adapted to provide white light during a brief period when immediate and delayed fluorescent imaging is not occurring so that a white light reflectance image can be captured as a background image. In yet another embodiment, while the illuminator 102, 152 provides a similar white light pulse, the light from the tissue is temporarily diverted to a second camera so that a white light reflectance image can be captured during a brief period when immediate and delayed fluorescent imaging is not occurring. Similarly, a dedicated background camera 199, shown in FIG. 1C, may be used to capture a white light reflectance image.

[0059] In embodiments where a reflectance "background" image is captured, the reflectance image is displayed adjacent to the delayed to immediate fluorescence ratio image. In an alternative embodiment, the delayed to immediate fluorescence ratio image is color encoded and overlaid on the reflectance image to allow direct identification of hypoxic tissue.

[0060] In an embodiment, a calibration is performed for each tissue type expected to be imaged, allowing quantification of oxygen levels in those tissue types.

[0061] Exemplary embodiments An experimental exemplary embodiment was tested as follows.

[0062] Nude female mice were subcutaneously injected with 106 human pancreatic adenocarcinoma BxPC3 cells in the flank area. After a growth period of approximately 3 weeks, when the tumors reached a diameter of approximately 8 mm, animals were selected for imaging. These nude mice were used in the imaging experiments described below.

[0063] ALA was either injected intraperitoneally (250 mg / kg) 6 h before imaging or Ameluz (trademark of Biofrontera AG, Leverkusen, Germany), a 10% ALA-containing ointment, was applied topically to the area of ​​interest 3 h before imaging, as described further below.

[0064] Images of tumors in live mice were acquired with a highly sensitive image intensified CMOS camera synchronized with a modulated diode laser of 635 nm with an average power of 50 mW. This laser provides pulsed fluorescence excitation light using 20 microsecond (μs) pulses at a frequency of 500 Hertz (Hz). The intensified CMOS camera was equipped with a bandpass filter of 697 ± 37 nanometers (nm), optical density (OD) 6 to remove any extraneous laser radiation interfering with the detection spectral window. The laser was partially focused to illuminate an area of ​​8 cm in diameter, resulting in a time-averaged illumination intensity at the sample of 500 microwatts per square centimeter (μW / cm). 2 The illumination intensity was measured with a power meter equipped with a photodiode power sensor.

[0065] The intensifier of the intensifier CMOS camera was directly controlled by a circuit synchronized with the laser pulse. The immediate fluorescence images were captured using an intensifier gate pulse width of 100 nanoseconds (ns) starting 10 μs after the rising edge of the laser pulse, while the delayed fluorescence images were captured using an intensifier gate pulse width of 1975 μs starting 2 μs after the end of the laser pulse. In each image frame, the immediate and delayed fluorescence were alternately integrated for 50 laser pulse gate periods in even and odd frames, respectively. We note that the immediate and delayed fluorescence signals are in the same emission band and therefore have matching spectra and instrument responses. Thus, the immediate and delayed fluorescence images of PPIX were acquired sequentially at an effective frame rate of 10 frames per second (fps), allowing for the “real-time” reconstruction of a normalized hypoxia image (delayed fluorescence / immediate fluorescence). Both the immediate and delayed fluorescence images were acquired with 2 × 2 pixel binning, resulting in a final image size of 800 × 600 pixels for further processing.

[0066] Immediate and delayed fluorescence images were background subtracted and spatially median filtered to remove hot pixels and readout noise, and time plots were smoothed using a 10-element sliding average filter.

[0067] The intensity of the delayed fluorescence signal is inversely correlated with the intracellular oxygen level. Therefore, imaging of PpIX delayed fluorescence can identify hypoxic regions, such as tumors. The ability to restore oxygen metabolism in real time is crucial for live monitoring of tumor resection during surgery.

[0068] To demonstrate the system's ability to measure oxygen fluctuations in vivo, initial experiments were performed using a 4 cm 2ALA was applied topically as Ameluz to the area of ​​​​the PPIX. Anesthetized mice were delivered an excessive CO2 pulse, causing rapid hypoxia between normal oxygen breathing. The variation of the PPIX signal was measured, which showed an inverse correlation between PPIX delayed fluorescence and in vivo pO2, as shown in Figure 5. Administration of a low dose of carbon dioxide to anesthetized mice showed a small response in delayed fluorescence and the ratio of delayed fluorescence to immediate fluorescence. On the other hand, administration of a large but not lethal dose of carbon dioxide showed a large response in delayed fluorescence and the ratio of delayed fluorescence to immediate fluorescence. As part of this initial calibration, both immediate fluorescence (IF) and delayed fluorescence (DF) signals were measured. The immediate fluorescence had minimal dependence on ambient oxygen. Therefore, we reasoned that by measuring the ratio R=DF / IF, we could correct for differences in PpIX concentration between tissues and tissue pigmentation without errors caused by baseline offsets or differences in wavelength filtering. The change in instantaneous fluorescence intensity with oxygen levels is minimal compared to the change in delayed fluorescence, so the instantaneous fluorescence appears to be constant compared to the delayed fluorescence. We also note that the distribution of PpIX (corresponding to the instantaneous fluorescence distribution) precisely coincides with the skin area where ALA was applied.

[0069] While this approach does not necessarily provide absolute measurements, it does allow the ability to monitor oxygen transients and spatial distribution within the tissue.

[0070] Most tumors exhibit chronic and / or cyclic hypoxia in microregions. Pancreatic tumors, in particular, are interstitial with few blood vessels, so hypoxia is often already present. Also, due to their structure, pancreatic tumors do not generate significant amounts of PpIX by themselves; rather, PpIX is generated systemically and distributed by the blood to both tumors and surrounding normal tissues. PpIX then accumulates in tumor tissues due to enhanced permeability and retention effects. Nude mice bearing subcutaneous human pancreatic adenocarcinoma BxPC3 tumors were used for imaging, and imaging was performed 6 hours after intraperitoneal injection of ALA (250 mg / kg). This extended time course was chosen to allow for the generation of PpIX in the mouse's entire body and its subsequent accumulation in the tumor. Some specimens analyzed in this study naturally exhibited sufficient contrast between tumor and normal tissues, as shown by the prepalpation elevation of the tumor DF / IF ratio of 602 relative to the normal tissue DF / IF ratio of 604 in Figure 6. The tissue was then palpated (606) prior to imaging, temporarily further enhancing the hypoxic state and significantly increasing the DF / IF ratio of the tumor compared to that of the normal tissue (608). As shown in Figures 6 and 7, the DF / IF ratio of the tumor took much longer to recover than that of the normal tissue. Due to poor vascularization, tumors are slow to reoxygenate, unlike normal tissues, which have relatively high blood pressure and relatively stable blood flow. This palpation-induced deoxygenation presented excellent contrast for up to 5 minutes, according to our experimental results based on the difference in kinetics shown in Figure 6. Before applying pressure to the tissue, the DF intensity level of PpIX is quite low. When pressure is applied, the blood is pushed out, causing the tissue to become deoxygenated and emit a strong DF signal as shown in Figure 6. Approximately one minute after palpation, the normal tissue recovers to normal pO2 and the tumor leaves a high contrast as shown in Figure 7. In addition, the temporal change in the DF / IF map ratio is parameterized for each pixel to display the kinetics of oxygen diffusion after palpation.

[0071] For images in Figures 7 and 8A-D, the fluorescent images were semi-transparently overlaid on the corresponding white light images using a custom-made color map. The reoxygenation rate constant maps in Figure 7 were calculated in MATLAB® by fitting the temporal image stacks of DF images to every 5 x 5 binned pixel using a single exponential decay function and least-squares regression method. The fitting was performed on background-subtracted images using the following equation: FiT =Ie -τ.time (1) where I and τ correspond to the fitted parameters, respectively, and τ is the rate constant displayed in Figure 7.

[0072] Figures 8A-8D show the DF / IF ratio over time, where the DF / IF ratio is displayed in color overlaid on a black and white image of the mouse's back. Figure 8A also shows the DF / IF ratio before palpation. Figure 8B shows the elevated DF / IF ratio 5 seconds after palpation, Figure 8C shows the elevated DF / IF ratio 30 seconds after palpation, and Figure 8D shows that the DF / IF ratio of the tumor tissue is still significantly elevated 70 seconds after palpation. Figure 9 shows a white light image of another anesthetized nude mouse overlaid with a DF / IF ratio image highlighting a human pancreatic tumor growing under the mouse's skin.

[0073] Because the immediate fluorescence signal shows a higher signal in the tumor underneath than in the mouse skin, PpIX immediate fluorescence may result in poor tumor-to-background contrast when imaging skin or subcutaneous tumors. Considering delayed fluorescence instead dramatically increases the contrast between tumor and normal tissue at the same time point. Although the raw DF data also show excellent contrast, considering the DF / IF ratio improves the contrast and allows correction for PpIX concentration and tissue color. The ratio R map is found to be more uniform throughout the tumor than the DF signal map. To quantify the contrast, the intensity normalized to the normal tissue average intensity for each image is shown in Figure 10. IF shows similar signals in healthy and normal tissues, while DF and R provide strong contrast. In this study, high contrast (5-fold in some cases) was demonstrated when comparing surrounding normal oxygenated tissue in a pancreatic cancer model. In addition, palpation of the tissue amplifies the signal, providing intuitive temporal contrast. This new contrast mechanism combines the necessary key technologies, including a pulsed laser, a highly sensitive high-speed time-gated synchronous fluorescence camera, and ALA to induce endogenous PpIX. This concept has direct application for human use and may be readily utilized as an intrinsic contrast mechanism to guide oncological surgery in the future.

[0074] We believe that it may be possible to calibrate this system using other oxygen reference indicators, such as palladium porphyrin optical probes, to provide absolute tissue oxygen levels. We also believe that shorter fluorescence excitation wavelengths could be used to reduce interference from deeper tissues. Using several excitation wavelengths and further image processing, it may also be possible to create a 3D map of tissue oxygenation.

[0075] combination It is anticipated that the features of the various embodiments described herein may be implemented in various combinations. Among the anticipated combinations are the following:

[0076] 1. A system designated as A for imaging oxygen concentration in a diffusion medium using a protoporphyrin IX probe, comprising: a pulsed light source operable at an excitation wavelength of a protoporphyrin IX (PPIX) fluorophore in the diffusion medium and configured to irradiate the diffusion medium with a light pulse; a gated electronic camera synchronized with the pulsed light source and configured to capture a delayed fluorescence image of the diffusion medium in at least one delayed fluorescence time window beginning after an end of a light pulse from the pulsed light source and not overlapping with a light pulse from the pulsed light source, the gated electronic camera further configured to capture an instantaneous fluorescence image of the diffusion medium in an instantaneous fluorescence time window that overlaps with a pulse of light pulse from the pulsed light source; a filter device disposed between the diffusion medium and the gated electronic camera, the filter device configured to pass fluorescent emission of PPIX while blocking light at an excitation wavelength of PPIX; and an image processor coupled to receive the instantaneous fluorescence image and the delayed fluorescence image from the gated electronic camera, the image processor configured to generate an image indicative of oxygenation of the diffusion medium based on at least the delayed fluorescence image.

[0077] A system designated AA, including a system designated A, wherein the image indicating oxygenation of the diffusion medium comprises an image representing a ratio of the delayed fluorescence image to the instant fluorescence image.

[0078] A system designated AB, including systems designated A or AA, wherein the pulsed light source comprises a structured light modulator adaptable to provide multiple spatially modulated light patterns at multiple spatial frequencies and phase offsets, the gated electronic camera is configured to acquire a structured light image of the diffusing medium illuminated by the spatially modulated light patterns, and the image processor is further configured to extract optical parameters from the structured light image.

[0079] A system designated AC, including systems designated A, AA, or AB, wherein the image processor is further configured to correct the delayed fluorescence image using the extracted optical parameters.

[0080] A method designated as B for generating an oxygen level map image of a diffusion medium, comprising: using an illuminator configured to provide a pulse of fluorescence excitation light; acquiring an instantaneous fluorescence image of the diffusion medium and a delayed fluorescence image of the diffusion medium using a gated camera, the instantaneous fluorescence image being acquired during the fluorescence excitation light pulse and the delayed fluorescence image being acquired after the fluorescence excitation light pulse and non-overlapping; and determining a ratio image from the delayed fluorescence image and the instantaneous fluorescence image, the ratio image providing an oxygen level image of the diffusion medium.

[0081] The method designated BA, including the method designated B, wherein determining the oxygen signal level further comprises performing a calibration.

[0082] The method designated BB, including the method designated B or BA, wherein the diffusion medium is mammalian tissue and an oxygen level image of the diffusion medium is used to guide surgical treatment of tissue in a subject or to diagnose a medical condition in the subject.

[0083] The method designated BC, including the method designated BB, wherein the oxygen level image of the diffusion media is used in combination with a perfusion image of the tissue to guide surgical resection of hypoxic tumor tissue.

[0084] The method designated BD, including the method designated BB, wherein the tissue comprises a tumor.

[0085] The method designated as BE, including the method designated as BC, wherein the tissue with low oxygenation comprises a tumor.

[0086] A method designated Bf, including methods designated B or BA, wherein the diffusion medium is mammalian tissue, and further comprising subjecting the tissue to a temporary state of hypoxia by applying pressure to the tissue. The method designated BG, including methods designated B, BA, or BF, wherein the diffusion medium is tissue of a living mammal, and further comprising administering aminolevulinic acid (ALA) to the living mammal.

[0087] A system designated AD, including systems designated A, AA, AB, or AC, wherein the pulsed light source is configurable to provide fluorescence stimulation light for a second fluorophore and the gated electronic camera is configurable to acquire real-time fluorescence images of the second fluorophore, the second fluorophore being configured to image tissue perfusion.

[0088] The method designated BH, including the methods designated B, BA, BB, BC, BD, BE, BF, or BG, wherein the delayed fluorescence image of the diffusion medium represents endogenous oxygen-sensitive fluorophores in mammalian tissue.

[0089] The method designated BJ, including the method designated BG of B, BA, BB, BC, BD, BE, BF, or BH, wherein the endogenous oxygen-sensitive fluorophore comprises PPIX.

[0090] A system designated BK, including a method designated B, BA, BB, BC, BD, BE, BF, BG, or BH, wherein the image sensor of the gated electronic camera provides an integration of delayed fluorescence light over multiple pulses of excitation light.

[0091] The method designated BL, including the methods designated B, BA, BC, BD, BE, BF, BG, BH, BJ, or BK, further comprising acquiring a reflectance image and providing a display selected from the group consisting of: the reflectance image adjacent to a ratio of delayed to instant fluorescence image; and the ratio of delayed to instant fluorescence image overlaid on the reflectance image.

[0092] The method designated BM, including the methods designated B, BA, BB, BC, BD, BE, BF, BG, BH, BJ, BK, or BL, wherein the immediate fluorescence image and the delayed fluorescence image are captured by separate image sensors. The method designated BN, including the methods designated B, BA, BB, BC, BD, BE, BF, BG, BH, BJ, BK, BL, or BM, further comprising overlaying an oxygen level image of the diffusion medium in color on a white light image of the diffusion medium.

[0093] Changes may be made to the above methods and systems without departing from the scope of this specification. Accordingly, the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not limiting. The following claims are intended to cover all the general and specific features described herein, as well as all statements of the scope of the methods and systems herein, and may lie therebetween as a matter of language.

Claims

1. 1. A system for imaging oxygen concentration in a diffusion medium using a protoporphyrin IX probe, comprising: a pulsed light source operable at an excitation wavelength of a protoporphyrin IX (PPIX) fluorophore in the diffusion medium and configured to illuminate the diffusion medium with a pulse of light; a gated electronic camera synchronized with the pulsed light source and configured to capture a delayed fluorescence image of the diffusion medium in at least one delayed fluorescence time window beginning after an end of a light pulse from the pulsed light source and not overlapping with a light pulse from the pulsed light source, the gated electronic camera further configured to capture an instantaneous fluorescence image of the diffusion medium in an instantaneous fluorescence time window overlapping with a pulse of a light pulse from the pulsed light source; a filter device disposed between the diffusion medium and the gated electronic camera, the filter device configured to pass the fluorescent emission of PPIX while blocking light at an excitation wavelength of PPIX; an image processor coupled to receive the instant fluorescence image and the delayed fluorescence image from the gated electronic camera, the image processor configured to generate an image indicative of oxygenation of the diffusion medium based on at least the delayed fluorescence image.

2. The system of claim 1 , wherein the image indicative of oxygenation of the diffusion medium comprises an image representing a ratio of the delayed fluorescence image to the instantaneous fluorescence image.

3. 2. The system of claim 1, wherein the pulsed light source comprises a structured light modulator adaptable to provide multiple spatially modulated light patterns at multiple spatial frequencies and phase offsets, the gated electronic camera is configured to acquire a structured light image of the diffusing medium illuminated by the spatially modulated light patterns, and the image processor is further configured to extract optical parameters from the structured light image.

4. The system of claim 3 , wherein the image processor is further configured to correct the delayed fluorescence image using the extracted optical parameters.

5. 1. A method for generating an oxygen level map image of a diffusion medium, comprising: using an illuminator configured to provide pulses of fluorescence excitation light; acquiring an immediate fluorescence image of the diffusion medium and a delayed fluorescence image of the diffusion medium using a gated camera, the immediate fluorescence image being acquired during a fluorescence excitation light pulse and the delayed fluorescence image being acquired after, and non-overlapping with, the fluorescence excitation light pulse; determining a ratio image from the delayed fluorescence image and the instantaneous fluorescence image, the ratio image providing an oxygen level image of the diffusion medium.

6. The method of claim 5 , wherein determining the oxygen signal level further comprises performing a calibration.

7. 6. The method of claim 5, wherein the diffusion medium is mammalian tissue and the oxygen level image of the diffusion medium is used to guide surgical treatment of tissue in a subject or to diagnose a medical condition of the subject.

8. The method of claim 7 , wherein the diffusion media oxygen level image is used in combination with the tissue perfusion image to guide surgical resection of hypoxic tumor tissue.

9. The method of claim 7 , wherein the tissue comprises a tumor.

10. The method of claim 8 , wherein the tissue with low oxygenation levels comprises a tumor.

11. 7. The method of claim 5 or 6, wherein the diffusion medium is mammalian tissue, further comprising subjecting the tissue to temporary hypoxia by applying pressure to the tissue.

12. 7. The method of claim 5 or 6, wherein the diffusion medium is tissue of a living mammal, and further comprising administering aminolevulinic acid (ALA) to the living mammal.

13. 2. The system of claim 1, wherein the pulsed light source is configurable to provide fluorescence stimulation light for a second fluorophore and the gated electronic camera is configurable to acquire real-time fluorescence images of the second fluorophore, the second fluorophore being configured to image tissue perfusion.

14. The method of claim 5 , wherein the delayed fluorescence image of the diffusion medium represents an endogenous oxygen-sensitive fluorophore of the tissue.

15. The method of claim 14 , wherein the endogenous oxygen-sensitive fluorophore comprises PPIX.

16. The system of claim 1 , wherein the image sensor of the gated electronic camera provides an integration of delayed fluorescent light over multiple pulses of excitation light.

17. 6. The method of claim 5, further comprising acquiring a reflectance image and providing a display selected from the group consisting of: the reflectance image adjacent to a ratio image of delayed to instantaneous fluorescence, and a ratio image of delayed to instantaneous fluorescence overlaid on the reflectance image.

18. The method of claim 5 , wherein the immediate fluorescence image and the delayed fluorescence image are captured by separate image sensors.

19. The method of claim 5 , further comprising overlaying an oxygen level image of the diffusion media in color onto the white light image of the diffusion media.

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