WO2015164774A1

WO2015164774A1 - Fluorescence guided surgical systems and methods gated on ambient light

Info

Publication number: WO2015164774A1
Application number: PCT/US2015/027564
Authority: WO
Inventors: Kristian SEXTON; Brian William Pogue; Christopher SCHAEF; Scott Christian DAVIS; Yan Zhao; Fadi EL-GHUSSEIN
Original assignee: The Trustees Of Dartmouth College
Priority date: 2014-04-25
Filing date: 2015-04-24
Publication date: 2015-10-29

Abstract

A fluorescence guided surgical system gated on ambient light includes a fluorescence imaging system for imaging a fluorescing agent in a subject during surgery on the subject, and a sensor for detecting periods of low brightness of the ambient light to activate the imaging only during the periods of low brightness. An ambient light gated method for generating fluorescence images to guide a surgical procedure includes detecting periods of low brightness of ambient light, and imaging a fluorescing agent in a subject undergoing surgery during a first subset of the periods of low brightness of the ambient light.

Description

FLUORESCENCE GUIDED SURGICAL SYSTEMS AND METHODS GATED ON

AMBIENT LIGHT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority from U.S.

Provisional Application Serial No. 61/984,402 filed April 25, 2014, which is incorporated herein by reference in its entirety.

U.S. GOVERNMENT RIGHTS

[0002] This invention was made with Government support under NIH Grant Nos. R01 CA 109558 and P01 CA084203, both awarded by the National Institutes of Health, and Department of Defense award W81XWH-09- 1-0661. The Government has certain rights in this invention.

BACKGROUND

[0003] Image guided surgery is used to guide surgical procedures, especially in situations where accuracy of the procedure is critical for a successful outcome. Image guided surgery provides the surgeon with a live video stream of the area of interest for the procedure. Procedures utilizing image guided surgery include removal of brain tumors and other procedures that are required to be minimally invasive. Fluorescence image guided surgery is an extension of image guided surgery. In fluorescence image guided surgery, the patient is administered a fluorescing agent or a precursor to a fluorescing agent, which preferably binds to certain types of tissue. Thus, the fluorescence images may provide distinction between different types of tissue. The primary application of fluorescence image guided surgery is in the removal of brain tumors, where the fluorescing agent preferably binds to the brain tumor, or the precursor agent is preferably metabolized into a fluorescing agent by the tumor. In this case, the surgeon utilizes a live video of fluorescence images to accurately remove as much of the tumor as possible with minimal damage to the surrounding healthy tissue. Fluorescence image guided surgery is currently undergoing clinical tests for use in a number of surgical oncology procedures.

SUMMARY

[0004] In an embodiment, a fluorescence guided surgical system gated on ambient light includes a fluorescence imaging system for imaging a fluorescing agent in a subject during surgery on the subject, and a sensor for detecting periods of low brightness of the ambient light to activate the imaging only during the periods of low brightness.

[0005] In an embodiment, an ambient light gated method for generating fluorescence images to guide a surgical procedure includes detecting periods of low brightness of ambient light, and imaging a fluorescing agent in a subject undergoing surgery during a first subset of the periods of low brightness of the ambient light.

[0006] In an embodiment, a fluorescence guided surgical system gated on ambient light includes a fluorescence detection system for detecting fluorescence from a fluorescing agent in a subject during surgery thereon, and a sensor for detecting periods of low brightness of the ambient light to activate the imaging only during the periods of low brightness.

[0007] In an embodiment, an ambient light gated method for generating fluorescence images to guide a surgical procedure includes (a) detecting periods of low brightness of ambient light, (b) inducing fluorescence in a subject undergoing surgery with fluorescence excitation light, and (c) measuring fluorescence of the subject during a first subset of the periods of low brightness of the ambient light.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing and other features and advantages of the disclosure will be apparent from the more particular description of embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different figures. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

[0009] FIG. 1 illustrates a fluorescence guided surgical system gated on ambient light, according to one embodiment.

[0010] FIG. 2 illustrates another fluorescence guided surgical system gated on ambient light, according to one embodiment.

[0011] FIG. 3 illustrates a method for generating ambient light gated fluorescence images to guide a surgical procedure, according to one embodiment.

[0012] FIG. 4 illustrates another method for generating ambient light gated fluorescence images to guide a surgical procedure, according to one embodiment.

[0013] FIG. 5 illustrates a timing sequence that may be used by the method of FIG. 4, according to one embodiment. [0014] FIG. 6 illustrates a method for generating ambient light subtracted fluorescence images, according to one embodiment.

[0015] FIGS. 7A and 7B show conceptual plots that illustrate the advantage of pulsed fluorescence imaging, according to one embodiment.

[0016] FIGS. 8A-8E show results of fluorescence imaging of tissue- simulating phantoms using (i) a conventional system and (ii) a pulsed fluorescence imaging system, according to one embodiment.

[0017] FIGS. 9A-9H show results of fluorescence imaging of mice brains using (i) a conventional system and (ii) a pulsed fluorescence imaging system, according to one embodiment.

[0018] FIGS. lOA-lOC show temporal and spectral properties of normal ambient light typical of a non-operating room setting.

[0019] FIG. 11 shows fluorescence images of tissue-simulating phantoms, and associated results, obtained under normal ambient lighting such as that illustrated in FIG. 10.

[0020] FIGS. 12A-12C show temporal and spectral properties of a tungsten halogen incandescent surgical lamp, according to one embodiment.

[0021] FIGS. 13A and 13B show minimal detectable concentrations of a fluorescing agent under tungsten halogen incandescent surgical lamp ambient light, according to one embodiment.

[0022] FIGS. 14A-14C show temporal and spectral properties of high intensity fluorescent lighting of an operating room, according to one embodiment.

[0023] FIGS. 15A and 15B show results of fluorescence imaging of tissue- simulating phantoms in the 700 nanometer channel without and with ambient light gating, respectively, under high intensity fluorescent lighting such as that illustrated in FIG. 14, according to one embodiment.

[0024] FIGS. 16A and 16B show results of fluorescence imaging of tissue- simulating phantoms in the 800 nanometer channel without and with ambient light gating, respectively, under high intensity fluorescent lighting such as that illustrated in FIG. 14, according to one embodiment.

[0025] FIGS. 17A-17C show simulated fluorescence imaging results obtained with and without ambient light gating, in the presence of high intensity ambient light from a fluorescent light source such as those used in an operating room, according to one embodiment.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The present disclosure is concerned with a major challenge of conventional fluorescence image guided surgery, namely that the surgical procedure must be performed in a dark or dimly lit environment in order to detect the fluorescence signal. While fluorescence images are useful for guiding the surgeon, the drawbacks associated with operating in a dark environment are significant.

[0027] Disclosed herein are fluorescence guided surgical (FGS) systems and methods that are compatible with performing the surgical procedure in a brightly lit environment, and therefore may facilitate more widespread adoption of fluorescence guided surgery. The FGS systems and methods utilize real-time detection of the ambient light brightness to activate fluorescence imaging only during periods of low ambient light brightness.

[0028] In the present disclosure, "low ambient light brightness" refers to ambient light brightness that is below a specified brightness threshold lower than the time-averaged ambient light brightness. "Minimal ambient light brightness" refers to the low ambient light brightness characteristic of the ambient light brightness in a time period around a local minimum, as a function of time, of the ambient light brightness. Thus, low ambient light brightness may refer to minimal ambient light brightness. In an example, minimal ambient light brightness refers to brightness below a certain threshold, such as brightness in the lower 20%, 10%, 5%, or 1% of the full brightness range.

[0029] In certain embodiments, the FGS systems and methods further take advantage of the regular brightness oscillation of typical operating room lighting.

Operating rooms are generally equipped with bright lights, the brightness of which undergo a periodic brightness variation at twice the utility frequency. For example, in a setting with a 60 Hertz (Hz) utility frequency, the brightness of the lights oscillates at a frequency of 120 Hz. The FGS systems and methods gate fluorescence imaging on the periods of low, or minimal, ambient light brightness. This greatly reduces the contribution to the fluorescence signal from the ambient light, especially in settings where the ambient light is provided by fluorescent light bulbs that tend to exhibit strong, periodic brightness variation. Accurate ambient light subtraction may be performed by capturing pairs of fluorescence images, with and without fluorescence excitation, at subsequent periods of low, or minimal, ambient light brightness. The FGS systems and methods are capable of capturing each image of such a pair at identical, or substantially identical, ambient light conditions, which ensures accurate ambient light subtraction.

[0030] Furthermore, as opposed to exposing the tissue undergoing surgery, to continuous fluorescence excitation illumination, the present FGS systems and methods use a pulsed light source to provide short and powerful pulses of light during the periods of minimal ambient light brightness. The short light pulses may be significantly more powerful than continuous illumination, while still staying below the safety-mandated limit for light exposure of the tissue. Hence, the signal-to-noise ratio, where the noise is predominantly caused by ambient light, is significantly greater for the pulsed systems and methods disclosed herein than for a conventional system relying on continuous fluorescence illumination excitation.

[0031] FIG. 1 illustrates one exemplary FGS system 100 gated on ambient light. FGS system 100 may be used to guide a surgeon 180 performing a surgical procedure on an area of a patient 170 in an environment lit by an ambient light source 190. The area of interest of patient 170, or portions of the area of interest, has been labeled with a fluorescing agent 175. FGS system 100 includes an ambient light sensor 110, a fluorescence imaging system 120, and a control module 130. Ambient light sensor 110 senses the brightness of ambient light emitted by ambient light source 190 and communicates brightness measurements to control module 130. Ambient light sensor 110 is, for example, a photodiode. Control module 130 gates operation of fluorescence imaging system 120 to activate fluorescence imaging system 120 during periods of low, or minimal, brightness of the ambient light emitted by ambient light source 190.

[0032] In one embodiment, ambient light sensor 1 10 includes circuitry for detecting periods of low, or minimal, brightness of the ambient light emitted by ambient light source 190. In another embodiment, ambient light sensor 110 communicates a continuous or regular signal to control module 130, which is indicative of the brightness of the ambient light emitted by ambient light source 190. In this embodiment, control module 130 includes circuitry that identifies periods of low ambient light brightness based upon the signal received from ambient light sensor 1 10.

[0033] Without departing from the scope hereof, ambient light sensor 110 may be replaced by a sensor that senses oscillations of the alternating current (AC) driving ambient light source 190 to detect when the current is zero or near zero. This sensor may be coupled to the utility line powering at least ambient light source 190 and sense the oscillations of the driving AC current through measurement of an electrical property. In one such example, this sensor is an inductive sensor. The sensor need not be coupled to a utility line powering only ambient light source 190, but may be coupled to a utility line that powers other systems as well. For example, the sensor may be coupled to the utility line powering the room wherein the surgical procedure takes place.

[0034] Optionally, FGS system 100 further includes a fluorescence image display 150 that displays fluorescence images and/or video to surgeon 180. Alternatively, FGS system 100 includes an interface that communicates fluorescence images and/or video captured by fluorescence imaging system 120 to a display external to FGS system 100. FGS system 100 may provide surgeon 180 with a real-time fluorescence image video.

[0035] FIG. 2 illustrates one exemplary FGS system 200, which is an embodiment of FGS system 100 (FIG. 1). FGS system 200 includes ambient light sensor 1 10 (FIG. 1), an illumination module 210, a camera 220, and a control module 230. Illumination module 210 and camera 220 together form an embodiment of fluorescence imaging system 120 of FIG. 1, and control module 230 is an embodiment of control module 130 of FIG. 1. Camera 220 captures fluorescence images of an area of interest 260 illuminated by illumination module 210. Area of interest 260 is, for example, an area of patient 170 (FIG. 1 ) labeled by fluorescing agent 175 (FIG. 1 ).

[0036] Ambient light sensor 110 senses brightness of ambient light, such as ambient light produced by ambient light source 190 (FIG. 1), and communicates measurements 270 thereof to control module 230. Generally, measurements 270 are signals indicative of measurement results, such as a voltage proportional to a measured ambient light brightness or an electrical pulse indicative of detection of low, or minimal, ambient light brightness. Control module 230 includes an interface 234 that receives measurements 270 from ambient light 110 and communicates control signals 272 and 274 to illumination module 210 and camera 220, respectively. Control module 230 further includes circuitry 232 that processes measurements 270 to generate control signals 272 and 274 therefrom. For example, circuitry 232 processes measurements 270 to identify periods of low, or minimal, ambient light brightness and generates control signals 272 and 274 for gating the operation of illumination module 210 and camera 220 to be active only during periods of low, or minimal, ambient light brightness. In an embodiment, circuitry 232 includes analog circuitry that processes measurements 270. In another embodiment, circuitry 232 includes a computing device, such as a processor coupled with a memory, or a microprocessor, that processes measurements 270. For example, circuitry 232 includes a digital data acquisition board incorporated into a computer and controlled using machine-readable instructions stored in non-volatile memory. The machine- readable instructions may be in the form of a Labview program.

[0037] In embodiments wherein ambient light sensor 110 includes circuitry for detecting periods of low, or minimal, ambient light brightness, ambient light sensor 110 communicates corresponding signals 270 to control module 230, which indicate detection of low, or minimal, ambient light brightness. In such embodiments, circuitry 232 is configured to process signals 270 to generate control signals 272 and 274 in accordance therewith. In embodiments wherein ambient light sensor 1 10 communicates a continuous or regular signal 270 to control module 130, indicative of the ambient light brightness, circuitry 232 is configured to process such a signal to identify periods of low, or minimal, ambient light brightness and generate control signals 272 and 274 in accordance therewith.

[0038] Illumination module 210 includes a light source 212 that generates fluorescence excitation light and an interface 218 for receiving control signal 272 from control module 230 via interface 234. Control signal 272 is, for example, a trigger signal causing emission of a pulse of fluorescence excitation illumination by light source 212, where the pulse duration is a preconfigured, or otherwise configurable, property of illumination module 210. In another example, control signal 272 includes a stop and a start signal for emission of a pulse of fluorescence excitation illumination by light source 212, where the stop and start signals define the pulse duration. Emission of fluorescence excitation illumination by light source 212 may be controlled by turning on and off the light generating component of light source 212, or by unblocking and blocking fluorescence excitation illumination generated by light source 212 using, for example, an electronically controlled shutter.

[0039] Optionally, illumination module 210 includes a focusing element 214 that focuses light emitted by light source 212 onto area of interest 260. Although not illustrated in FIG. 2, illumination module 210 may include an optical fiber, or other form of light guide, that delivers light from light source 212 to area of interest 260, without departing from the scope hereof. The output end of the optical fiber, or other form of light guide, may be coupled with focusing element 214 for focusing of fluorescence excitation illumination onto area of interest 260. Illumination module 210 may include a wavelength filter 216 that selects a desired spectral portion of illumination emitted by light source 212.

[0040] In certain embodiments, light source 212 is a light-emitting diode (LED). The LED may emit light in the range near 405 nanometers (nm), for example for efficient excitation of protoporphyrin IX (PpIX). In another example, the LED emits light in the range near 630 nm for excitation of fluorophores such as IRDye 680RD or those based upon Rhodamine, as well as excitation of PpIX. Longer wavelength excitation illumination presents an advantage in terms of penetration depth into tissue. Hence, as compared to an LED in the blue or violet portions of the spectrum, an LED in the red portion of the spectrum may enable improved identification of sub-surface unhealthy tissue that should be removed. In other embodiments, light source 212 is one or more of a laser, a halogen light bulb, a tungsten lamp, or any other light source suitable for generating fluorescence excitation emission.

[0041] Camera 220 includes an image sensor 222, a wavelength filter 224, and an interface 228. Wavelength filter 224 selects a desired spectral portion of light propagating towards image sensor 222, as known in the art of fluorescence imaging. Image sensor 222 captures fluorescence images of area of interest 260, wavelength filtered by wavelength filter 224 to reduce the contribution of other light than the fluorescence induced by illumination module 210. Camera 220 receives control signal 274 through interface 228, such that fluorescence images captured by camera 220 are gated on the ambient light brightness to be captured during periods of low, or minimal, ambient light brightness. In one example, control signal 274 triggers image capture by image sensor 222, where the exposure time is a preconfigured, or otherwise configurable, property of camera 220. In another example, control signal 274 triggers image capture by image sensor 222 as well as the associated exposure time. In one embodiment, image sensor 222 is an intensified charge-coupled device (ICCD) image sensor. In another embodiment, image sensor 222 is an electron-multiplied charge-coupled device

(EMCCD) image sensor, a charge-coupled device (CCD) image sensor, or a

complementary metal oxide semiconductor (CMOS) image sensor. In certain

embodiments, camera 220 is capable of performing measurements at high temporal resolution, which provides consistent timing of fluorescence image capture with respect to ambient light variations. For example, camera 220 is capable of capturing images with timing resolution of at least 50 kiloHertz.

[0042] In embodiments of FGS system 200 that include wavelength filter 216, wavelength filters 224 and 216 are advantageously configured to select mutually exclusive spectral portions, such that the direct, non-fluorescence related contribution from light emitted by illumination module 210 to the fluorescence images is minimized. In an embodiment, wavelength filter 224 includes a plurality of different wavelength filters mounted in a filter switching device, such as a filter wheel. This embodiment may be utilized to perform multispectral imaging of area of interest 260, in a scenario where area of interest 260 includes two or more different fluorescing agents. Control module 230 may control the filter switching device through interfaces 234 and 228. Alternatively, wavelength filter 224 may be a liquid crystal tunable filter controlled by control module 230 to provide multispectral imaging. Additionally, light source 212 may include several different light sources, having different wavelength ranges, for multispectral fluorescence imaging.

[0043] Fluorescence images captured by camera 220 are outputted through interface 228. In an embodiment, FGS system 200 includes fluorescence image display 150 (FIG. 1 ) that displays fluorescence images captured by camera 220 and received from interface 228.

[0044] In an embodiment, FGS system 200 further includes an analysis module 250 that processes fluorescence images generated by camera 220. Analysis module 250 includes a processor 252 and a memory 254. Memory 254 includes instructions 256 and, optionally, a data storage 258 for storing data such as fluorescence images. Analysis module 250 receives fluorescence images from interface 228 of camera 220. Analysis module 250 may process fluorescence images captured by camera 220 and communicate processed images, and/or non-image data generated from the fluorescence images captured by camera 220, to fluorescence image display 150. As will be discussed further in connection with FIG. 6, analysis module 250 may be configured to generate ambient light subtracted fluorescence images from fluorescence images captured by camera 220. Analysis module 250 may communicate such ambient light subtracted fluorescence images to fluorescence image display 150 for displaying thereon. [0045] Without departing from the scope hereof, camera 220 may be replaced by, or function in conjunction with, a non-imaging fluorescence detector, such as a photodiode or photomultiplier tube, which measures total fluorescence received from area of interest 260. Additionally, although potentially less advantageous from a signal-to- noise perspective, illumination module 210 may continuously illuminate area of interest 260, while camera 220 is gated based on ambient light brightness, as discussed above, without departing from the scope hereof.

[0046] FIG. 3 illustrates one exemplary method 300 for generating ambient light gated fluorescence images to guide a surgical procedure on an area of interest of a subject that includes a fluorescing agent, such as a portion of patient 170 (FIG. 1) including fluorescing agent 175 (FIG. 1). Method 300 is performed, for example, by FGS system 200 of FIG. 2 or FGS system 100 of FIG. 1.

[0047] In a step 310, the brightness of ambient light is sensed to detect periods of low, or minimal, ambient light brightness. In one example of step 310, ambient light sensor 1 10 (FIGS. 1 and 2) senses the brightness of ambient light, such as light emitted by ambient light source 190 (FIG. 1). Ambient light sensor 110 may cooperate with control module 130 (FIG. 1) to detect periods of low, or minimal, ambient light brightness. In an embodiment, the detected periods of low, or minimal, ambient light brightness, are periods of identical, or substantially identical, ambient light brightness. This embodiment facilitates accurate background subtraction, as discussed below in connection with FIG. 6. For example, ambient light sensor 110 (FIG. 1) cooperates with control module 130 (FIG. 1) to detect when the ambient light brightness drops below a specified threshold, or is within a range defined by an upper and a lower threshold. In an embodiment, not illustrated in FIG. 3, the periods detected in step 310, and utilized below in step 320, are not necessarily periods of low ambient light brightness, but rather periods of identical, or substantially identical, brightness. This embodiment also facilitates improved background subtraction, as compared to methods that do not synchronize the fluorescence imaging with the ambient light brightness variation.

[0048] In a step 320, fluorescence imaging of a fluorescing agent in a subject is performed during periods of low, or minimal, brightness. In one example of step 320, fluorescence imaging system 120 (FIG. 1) performs fluorescence imaging of fluorescing agent 175 (FIG. 1) in patient 170 (FIG. 1), where the fluorescence imaging is gated by control module 130 (FIG. 1) according to the detection of periods of low, or minimal, brightness performed in step 310.

[0049] In one embodiment, step 320 is performed concurrently with step 310, such that step 310 monitors ambient light brightness continuously or regularly while step 320 performs fluorescence imaging during at least some of the periods of low, or minimal, ambient light brightness. In another embodiment, steps 310 and 320 are performed alternatingly. Upon detection of a period of low, or minimal, ambient light brightness in step 310, method 300 proceeds to perform step 320. Upon completion of a fluorescence measurement, in step 320, associated with the period of low, or minimal, ambient light brightness in step 310, method 300 returns to perform step 310 and continues to perform steps 310 and 320 in this alternating fashion.

[0050] Step 320 includes steps 322 and 324. In step 322, fluorescence of the fluorescing agent is induced. In one example of step 322, illumination module 210 (FIG. 2) delivers fluorescence excitation illumination to area of interest 260 (FIG. 2). In step 324, fluorescence images of the subject are captured during a first subset of the periods of low, or minimal, brightness detected in step 310. In one example of step 324, camera 220 (FIG. 2) captures images of area of interest 260 (FIG. 2), wherein the image capture is gated by control signal 274 (FIG. 2) received from control module 230 (FIG. 2). The first subset may be all detected periods of low, or minimal, ambient light brightness, or a subset thereof such as every second or fourth detected period of low, or minimal, ambient light brightness. Step 322 may induce fluorescence continuously or induce fluorescence only when required by step 324. In an embodiment, step 324 is performed with high timing resolution to accurately synchronize image capture with the periods of low, or minimal, ambient light brightness detected in step 310. For example, step 324 is performed with timing resolution of at least 50 kiloHertz.

[0051] In certain embodiments of method 300, the periods of low, or minimal, brightness are periods of minimal brightness, where the duration of the periods are minimized to minimize the relative contribution from ambient light to the fluorescence images captured in step 320. The optimal duration of the periods is a tradeoff between minimizing the ambient light contribution and maintaining sufficient fluorescence signal, integrated over one or more of the periods. In practical examples, the periods of minimal brightness may be periods where the ambient light brightness is in the lower 1 %, 5%, 10%, or 20% of the full brightness variation range. [0052] In an optional step 330, the fluorescence images captured in step 320 are outputted. In one example of step 330, camera 220 communicates captured fluorescence images to analysis module 250 (FIG. 2) and/or fluorescence image display 150 (FIGS. 1 and 2). Method 300 may perform step 330 as fluorescence images become available from step 320, for example to produce a real time video stream of fluorescence images.

[0053] Optionally, method 300 further includes a step 301 of generating ambient light with a periodic brightness variation. In one example of step 301, ambient light with a periodic brightness variation is generated using ambient light source 190 (FIG. 1). Although not indicated in FIG. 3, optional step 301 is performed concurrently with steps 310 and 320.

[0054] Ambient light may originate from several different sources, without departing from the scope hereof. For example, method 300 is performed in an

environment that is lit by ambient light having a periodic brightness variation, as prescribed in step 301, as well as by near-constant ambient light from light sources, such as daylight and incandescent light bulbs. However, the greatest benefit of method 300, as compared to methods not utilizing gating on ambient light, is achieved in environments predominantly lit by ambient light having significant brightness variation. FGS systems 100 and 200 of FIGS. 1 and 2, respectively, may perform method 300 in the presence of ambient light originating from several different sources.

[0055] Similar to the discussion in connection with FIG. 2, method 300 may be modified to perfonn ambient light gated fluorescence detection, wherein the detection is a non-imaging fluorescence measurement such as measurement of a total amount of fluorescence, without departing from the scope hereof. In this case, step 324 is replaced by a step of non-imaging fluorescence detection, and optional step 330 is replaced by an optional step of outputting measurements resulting therefrom.

[0056] FIG. 4 illustrates one exemplary method 400 for generating ambient light gated fluorescence images to guide a surgical procedure on an area of interest of a subject that includes a fluorescing agent, such as a portion of patient 170 (FIG. 1) including fluorescing agent 175 (FIG. 1). In method 400, fluorescence excitation and fluorescence imaging are gated on the ambient light brightness. Method 400 is performed, for example, by FGS system 200 of FIG. 2 or FGS system 100 of FIG. 1. Method 400 is an embodiment of method 300 of FIG. 3. [0057] In a step 410, the ambient light brightness is sensed. In one example of step 410, ambient light sensor 110 (FIGS. 1 and 2) senses the brightness of ambient light such as light emitted by ambient light source 190 (FIG. 1) and/or other light sources. Step 410 includes a step 412 of detecting periods of low, or minimal, ambient light brightness. In one example of step 412, circuitry 232 of control module 230 (FIG. 2) processes measurements received from ambient light sensor 110 (FIGS. 1 and 2), through interface 234 (FIG. 2), to identify periods of low, or minimal, ambient light brightness. Step 410 is an embodiment of step 310 of method 300 (FIG. 3).

[0058] As discussed in connection with step 310 (FIG. 3), in an embodiment, step 410 detects periods of low, or minimal, ambient light brightness, that are periods of identical, or substantially identical, ambient light brightness. This embodiment facilitates accurate background subtraction, which is discussed further in connection with FIG. 6 below. In an embodiment, not illustrated in FIG. 4, the periods detected in step 410, and utilized below in step 430, are not necessarily periods of low ambient light brightness, but rather periods of identical, or substantially identical, brightness. This embodiment also facilitates improved background subtraction, as compared to methods that do not synchronize the fluorescence imaging with the ambient light brightness variation.

[0059] In certain embodiments, the periods of low, or minimal, brightness are periods of minimal brightness, where the duration of the periods are minimized to minimize the relative contribution from ambient light to the fluorescence images captured in step 430. In this embodiment, the optimal duration of the periods is a tradeoff between minimizing the ambient light contribution and maintaining sufficient fluorescence signal, integrated over one or more of the periods. In practical examples hereof, the periods of minimal brightness may be periods where the ambient light brightness is in the lower 1%, 5%, 10%, or 20% of the full brightness variation range.

[0060] In a step 420, control signals are communicated to the fluorescence imaging system. The control signals gate the fluorescence imaging to occur during periods of low, or minimal, ambient light brightness, as detected in step 412. Step 420 includes steps 422 and 424. In step 424, a first control signal is communicated to the camera of the fluorescence imaging system to synchronize image capture with a first subset of the periods of low, or minimal, ambient light brightness detected in step 412. In one example of step 424, control module 230 (FIG. 2) communicates control signal 274 (FIG. 2) to camera 220 (FIG. 2) to synchronize image capture by image sensor 222 (FIG. 2) with the detected periods of low, or minimal, ambient light brightness. In step 422, a second control signal is communicated to the illumination module of the fluorescence imaging system to synchronize emission of fluorescence excitation illumination with a second subset of the periods of low, or minimal, ambient light brightness detected in step 412. In one example of step 422, control module 230 (FIG. 2) communicates control signal 272 (FIG. 2) to illumination module 212 (FIG. 2) to synchronize emission of fluorescence excitation illumination with the detected periods of low, or minimal, ambient light brightness. The first subset of periods may be all detected periods of low, or minimal, ambient light brightness, or a subset thereof such as every second or fourth detected period of low, or minimal, ambient light brightness. In an embodiment, the second subset of periods is a subset of the first subset of periods.

[0061] In one embodiment, the first and second control signals include individual features, where each individual feature originates from the detection of a period of low, or minimal, ambient light brightness in step 412. For example, the first and second control signals include a series of trigger pulses, where each trigger pulse is generated from an associated detection of a period of low, or minimal, ambient light brightness. In another embodiment, relevant in scenarios where the ambient light brightness exhibits periodic brightness variation, one or both of the first and second control signals may include a single feature, such as a single timing pulse. Since the ambient light brightness variation is periodic, this single feature synchronizes performance of steps 432 and 434 with the timing of periods of low, or minimal, ambient light brightness for a duration that includes several periods of low, or minimal, ambient light brightness.

[0062] In a step 430, fluorescence imaging is performed in accordance with the control signals received by the fluorescence imaging system in step 420. Step 430 includes steps 432 and 434. Steps 430, 432, and 434 are embodiments of steps 320, 322, and 324, respectively, of method 300 (FIG. 3). In step 432, fluorescence of the fluorescent agent in the area of interest is induced in accordance with the second control signal. In one example of step 432, illumination module 210 (FIG. 2) delivers fluorescence excitation illumination to area of interest 260 (FIG. 2) during periods of low, or minimal, ambient light brightness in accordance with control signal 272 (FIG. 2). In step 434, fluorescence images are captured in accordance with the first control signal. In one example of step 434, camera 220 (FIG. 2) captures fluorescence images of area of interest 260 (FIG. 2) during periods of low, or minimal, ambient light brightness according to control signal 274 (FIG. 2). In an embodiment, step 434 is performed with high timing resolution to accurately synchronize image capture with the periods of low, or minimal, ambient light brightness detected in step 410. For example, step 434 is performed with timing resolution of at least 50 kiloHertz.

[0063] In one embodiment, steps 420 and 430 are performed concurrently with step 410, such that step 410 monitors ambient light brightness continuously or regularly while steps 420 and 430 produce fluorescence images during at least some of the periods of low, or minimal, ambient light brightness. In another embodiment, step 410 is performed alternatingly with steps 420 and 430. Upon detection of a period of low, or minimal, ambient light brightness in step 412, method 400 proceeds to perform steps 420 and 430. Upon completion of a fluorescence measurement, in step 430, associated with the period of low, or minimal, ambient light brightness in step 412, method 400 returns to perform step 410 and continues in this fashion to perform step 410 alternatingly with steps 420 and 430.

[0064] In an optional step 440, method 400 performs step 330 of method 300, as discussed in connection with FIG. 3. Optionally, method 400 includes a step 401 of performing step 301 of method 300, as discussed in connection with FIG. 3.

[0065] Similar to the discussion in connection with FIG. 3, method 400 may be modified to perform ambient light gated fluorescence detection, wherein the detection is a non-imaging fluorescence measurement such as measurement of a total amount of fluorescence, without departing from the scope hereof. In this case, step 434 is replaced by a step of non-imaging fluorescence detection, and optional step 440 is replaced by a step of outputting measurements resulting therefrom.

[0066] FIG. 5 illustrates one exemplary timing sequence 500 for performing step 430 of method 400 (FIG. 4). When performed according to timing sequence 500, method 400 (FIG. 4) generates a series of fluorescence images alternating between (a) fluorescence images captured while inducing fluorescence by performing step 432 and (b) fluorescence images captured without inducing fluorescence. The latter images may be utilized for background subtraction.

[0067] Diagram 510 shows an exemplary, periodically varying ambient light brightness cycle 51 1, plotted as ambient light brightness 512 as a function of time 513. Ambient light brightness cycle 51 1 exhibits a series of periods 514 of minimal brightness. For clarity of illustration, only one period 514 of minimal brightness is indicated in diagram 510.

[0068] The first control signal of step 424 (FIG. 4) is formed such that fluorescence image capture of step 434 (FIG. 4) is performed during every other period 514 of minimal ambient light brightness, as indicated in diagram 520. Diagram 520 shows fluorescence image capture 523 as a function of time 513. Arrows 515 indicate the periods 514 of minimal ambient light brightness, for which a fluorescence image is captured. For clarity of illustration, only one arrow 515 is labeled in FIG. 5. Features 521 and 522 indicate periods during which an image sensor is exposed to capture the fluorescence image.

[0069] The second control signal of step 422 (FIG. 4) is formed such that the fluorescence excitation illumination of step 432 (FIG. 4) is delivered to the area of interest during every other period 514 of minimal ambient light brightness, during which a fluorescence image is captured, as indicated in diagram 530. Diagram 530 shows fluorescence excitation illumination 532 as a function of time 513. Arrows 525 indicate the periods 514 of minimal ambient light brightness, for which a fluorescence excitation illumination is delivered to the area of interest. For clarity of illustration, only one arrow 525 is labeled in FIG. 5. Fluorescence excitation illumination is delivered during periods 531, indicated in diagram 530. Accordingly, fluorescence images captured during periods 521 , indicated in diagram 520, include fluorescence induced by performing step 432 (FIG. 4). Fluorescence images captured during periods 522 do not include fluorescence induced by performing step 432 (FIG. 4). Signal present in the fluorescence images captured during periods 522 may be attributed to the ambient light.

[0070] In an example, periods 514 of minimal brightness occur at a rate of 120 Hz, which is typically the case when a fluorescent light is powered by a utility network operating at a utility frequency of 60 Hz. Thus, each of periods 521 and 522 occur at a rate of 30 Hz, such that each 30 Hz cycle produces two fluorescence images: one image that includes induced fluorescence and one image that does not include induced fluorescence. 30 Hz is a standard video rate. Therefore, fluorescence images captured during periods 521 or 522, or fluorescence images generated from processing fluorescence images captured during periods 521 and 522, may be displayed at a standard video rate. [0071] Although FIG. 5 shows periods 531 as having the same duration as periods 521, period 531 may have duration different from that of period 521 , without departing from the scope hereof. For example, period 531 may be slightly longer than period 521 to ensure that, even in the presence of some timing inaccuracy, fluorescence excitation illumination is present throughout fluorescence image capture. Furthermore, periods 522 may have duration different from periods 521, for example to allow longer exposure while capturing the fluorescence images associated with periods 522. In one example, periods 531 have duration less than two milliseconds (ms). In another example, periods 531 have duration in the range between 0.05 ms and 1 ms.

[0072] FIG. 6 illustrates one exemplary method 600 for generating ambient light subtracted fluorescence images. Method 600 includes an embodiment of method 400 (FIG. 4). In a step 610, method 600 performs method 400 with the second subset, in step 422, being a subset of the first subset in step 424. The second subset is every other period of low, or minimal, brightness included in the first subset. Hence, step 610 generates pairs < of fluorescence images Is and ½, where ¾ is captured while inducing fluorescence and IB is captured without inducing fluorescence. Is may be considered a "signal" image, while I_B may be considered a "background" image, and method 600 performs background subtraction. In one example of step 610, FGS system 200 (FIG. 2) performs method 400 according to timing sequence 500 (FIG. 5). In another example, each fluorescence image Is and IB of a pair of fluorescence images is captured during the same period of low, or minimal, ambient light brightness.

[0073] In a step 620, for each pair of fluorescence images Is and ¾, an ambient light subtracted fluorescence image ULS is generated by subtracting ¾ from Is. In situations where Is and ¾ are captured using different exposure times and/or camera gains, the subtraction may involve a scaling of one of the images. Since I_B is captured without inducing fluorescence, signal in ¾ may be attributed to ambient light. Is, on the other hand, is captured while inducing fluorescence, and Is therefore includes induced fluorescence as well as signal attributable to ambient light. Hence, IALS = IS-IB, with optional scaling if required, is an ambient light subtracted fluorescence image. In one example of step 620, processor 252 of analysis module 250 (FIG. 2) performs step 620 according to instructions 256 (FIG. 2). Processor 252 may retrieve fluorescence images Is and IB from data 258 (FIG. 2) or receive fluorescence images Is and I_B directly from camera 220 (FIG. 2). [0074] In an embodiment, fluorescence images Is and ¾ are captured during periods of identical, or substantially identical, ambient light brightness. This embodiment facilitates accurate background subtraction. Such periods may or may not be periods of low, or minimal, ambient light brightness. However, frequently the induced fluorescence signal in ¾ is small compared to the ambient light contribution. Therefore, utilizing periods of low, or minimal, ambient light brightness typically provides improved results, as compared to when utilizing periods of greater ambient light brightness. The accuracy of the ambient light subtraction obtained in step 620 is a function of the accuracy of the mutually respective timing of fluorescence image Is and ¾ within an image pair. Hence the accuracy of the ambient light subtraction may benefit from capturing fluorescence images Is and ¾ at high timing resolution, as discussed in connection with step 324 of method 300 (FIG. 3), such that fluorescence images Is and ¾ are captured under ambient light conditions that are as identical as possible.

[0075] In a step 630, ambient light subtracted fluorescence images IALS are outputted. Method 600 may perform step 630 as ambient light subtracted fluorescence images IALS become available from step 620 to produce a real time video of ambient light subtracted fluorescence images IALS- In one example of step 630, analysis module 250 (FIG. 2) outputs ambient light subtracted fluorescence images IALS to fluorescence image display 150 (FIGS. 1 and 2). Following the discussion in connection with FIG. 5, an example of method 600 provides a real time video of ambient light subtracted fluorescence images IALS at a standard video rate of 30 Hz.

[0076] Although not illustrated in FIG. 6, method 600 may include an additional step, after step 620, of averaging several ambient light subtracted images IALS, to improve the ambient light subtraction accuracy, without departing from the scope hereof.

[0077] Method 600 may be applied to non-imaging fluorescence detection and, for example, provide an ambient light subtracted total fluorescence signal, without departing from the scope hereof.

[0078] Methods 300 (FIG. 3), 400 (FIG. 4), and 600 (FIG. 6) are readily extended to provide multispectral fluorescence imaging. For example, different subsets of periods of low, or minimal, ambient light brightness are dedicated to capture of fluorescence images associated with different fluorophores. [0079] The ambient light gated fluorescence guided systems and methods disclosed herein may be applied to non-surgical medical procedures, without departing from the scope hereof. For example, the ambient light gated fluorescence guided systems and methods disclosed herein may be applied to fluorescence guided radiation therapy. Furthennore, the ambient light gated fluorescence guided systems and methods disclosed herein may be used in conjunction with conventional image guidance systems to provide a surgeon with both fluorescence images and conventional images.

Example I: Pulsed-light imaging for fluorescence guided surgery under normal room lighting

[0080] This Example describes an FGS system, developed specifically for imaging under room lights, which exploits pulsed excitation light and fluorescence imaging. The Example demonstrates the benefit of using pulsed excitation illumination and fluorescence image capture synchronized therewith, without the added benefit of synchronizing fluorescence imaging with ambient light variation. The system described in this Example is an embodiment of FGS system 200 (FIG. 2) implemented without ambient sensor 110 (FIGS. 1 and 2). This approach has been used to suppress signals for in vivo multispectral fluorescence imaging. Pulsed-light imaging can also increase the speed of image acquisition in surgical applications where excitation power is likely to dictate the minimum acquisition time. In the system presented here, the first realization is shown in the context of wide-field video-rate capable FGS imaging. The theoretical value of the instrument is highlighted and a direct comparison with an industry standard operating microscope is made using liquid tissue phantoms as well as in vivo studies.

[0081] The principle advantage of pulsed-light imaging is relatively simple; namely, that reducing acquisition time while maintaining the same radiant exposure reduces the contribution of ambient light in the signal. The effect in turn maximizes the dynamic range of the imaging system to the fluorescence signal and enables real time ambient light subtraction. It can be illustrated by considering the detected signal, Sd, in the presence of both the fluorescence excitation source, such as light source 212 (FIG. 2) and ambient light:

[0082] ¾ = ε^■ c^■ Φ^■ E_ex(_t)dt + / /^■ E_ex(t)dt + J E_A(t)dt,

[0083] where E_ex is the irradiance from the excitation source, E_Ais irradiance from ambient light, t is integration time, c, ε, and Φ are the concentration, molar extinction coefficient and quantum yield of the fluorophore, respectively, and / is some factor for nonspecific signal resulting from excitation light. FIG. 7A illustrates this principle by plotting 5_das a function of time for two systems, one which provides a fixed excitation irradiance and the other, a theoretical construct, which provides a fixed radiant exposure, H, where H = / E_ex(t)dt, both in the presence of a constant ambient light intensity.

[0084] FIGS. 7A and 7B are conceptual plots to illustrate the advantage of pulsed fluorescence imaging for low level fluorescence detection in the presence of high ambient lighting brightness. FIG. 7A shows detected signal 701 versus acquisition time 702 for a standard CCD where excitation light remains constant (curve 710), for an ICCD where excitation light increases proportionally as acquisition time decreases (curve 720), and for ambient light only with no excitation (curve 730). FIG. 7B shows background subtracted signal 703 versus acquisition time 704 for CCD (curve 750) and ICCD (curve 760) in the context of nonspecific background signal and noise for both fixed radiant exposure, H (shaded area 770 extending up to 100 ms acquisition time) and fixed irradiance, E (shaded area 780). Note that signal differences have been exaggerated to enable easy visualization.

[0085] In FIG. 7A, the detected signal includes the sum of contributions from fluorescence excitation illumination and ambient light that must remain below some maximum value for the system to avoid saturation (here chosen to be 16 bits). At the same time, the portion of the detected signal from the fluorophore must be sufficiently above the nonspecific background signal produced by excitation light to provide acceptable contrast-to-noise ratio. The contrast-to-noise ratio is defined as (signal from specified concentration - signal from control)/(standard deviation of pixel values within the control region of interest), where the control is a tissue-simulating phantom with no fluorophore. The fact that the nonspecific excitation background signal (generally a combination of excitation light leakage and nonspecific fluorescence) is a function of excitation power prevents an increase in excitation power from producing a directly proportional increase in contrast to noise. However, at the lowest fluorescent levels, the increased signal that results from a greater E_ex may mean the difference between detection and loss of the desired signal within the noise floor. The tradeoffs are evident in FIG. 7B, where both the background subtracted signals and the sum of nonspecific excitation background and noise for the two systems are plotted. The presence of strong ambient lighting prohibits the common solution of simply using longer imaging times to achieve adequate fluorescence signal. From a theoretical standpoint, the graph makes it clear that the optimum system provides just enough irradiance to remain just below saturation at the shortest possible acquisition times, which both maximizes fluorescence to nonspecific excitation background signal as well as minimizes imaging time. However, practical limits dictate the minimum exposure time of the camera as well as the maximum irradiance that the light source can provide.

[0086] As integration time decreases, it is critical to maintain adequate radiant exposure while also maintaining complying with safety-mandated maximum permissible exposure (MPE) limits. The American National Standard (ANSI) Z136.1-2000 specifies the MPE optical values for skin and eye. Given no published values specific to the surgical cavity, the skin limits could be used. The pulsed MPE values are the lowest of the single pulse, multipulse, and average power limit as described in the ANSI standards, where average exposure cannot exceed the 200 mW/cm² limit for continuous illumination. At pulse widths below 1000 8, frequencies of 10-100 Hz and imaging times on the order of hours, there is a distinct advantage to using pulsed light in regard to MPE. Pulsed light can deliver a considerably greater instantaneous power with the extent of the increase depending on the fraction of time that the tissue is exposed. For example, with an acquisition time of 500 μ≤, a pulse frequency of 50 Hz, and an imaging time of 1 hour, the MPE is dictated by the average power limit which allows each pulse to deliver up to 8000 mW/cm². This enables the exposure for each pulse to be ~40 times the power allowed during continuous light imaging. As a result, the practical limit of pulsed wide-field illumination is the power of the light source. As long as the necessary power can be delivered, the same fluorescent signals can be obtained at increasingly shorter gate widths, which is essential for both suppressing background signal and achieving rapid acquisition rates.

[0087] The pulsed-light system described here is configured to image protoporphyrin IX (PpIX) under surgical conditions. It consists of a PI-MAX 3-1024 ^χ 256 camera (26 μιη pixel size) (Princeton Instruments, Acton, Massachusetts) attached to an articulating arm via a custom mounting plate. The camera utilizes a UV Generation II intensified CCD (ICCD), which is able to achieve exposure times on the order of microseconds rather than the milliseconds typical of a standard CCD or electron multiplying CCD (EMCCD). Light received from the tissue is collected by a 24 mm F1.8 lens (Sigma, Ronkonkoma, New York) and then passes through an eight-position highspeed filter wheel (Edmund, Barrington, New Jersey) before focusing on the sensor. The setup enables efficient light collection from the lens and uses standard 1.0 in. (2.54 cm) diameter interference filters (here, a 700 nm filter with 40 nm FWHM from Omega, Brattleboro, Vermont) with the filter wheel providing the potential to image multiple fluorophores or perform multispectral imaging. Surrounding the lens is an excitation light positioning system capable of holding up to eight SpecBright pulsed LED area lights (ProPhotonix, Salem, New Hampshire) to illuminate the surgical field. In this study, four 630 nm LED's filtered with 1.0 in. 650 nm short pass filters (Edmund Optics, Barrington, New Jersey) were used. The LEDs provide wide field illumination with a reasonably narrow bandwidth and relatively high power, and in pulsed mode can be overdriven to 10 times the maximum power achievable in continuous operation (provided the pulse is below 1 ms and the duty cycle is less than 5%). From 25 cm, this four-light system running in pulsed mode is capable of providing 360 mW/cm² at the tissue surface. In the experiments described here the ICCD was allowed a 500 exposures and was synchronized to the LED pulses with all systems controlled through Labview (National Instruments, Austin TX). Dynamic background subtraction was performed in real time with each pulsed light acquisition being preceded by an equivalent acquisition in the absence of excitation light.

[0088] To compare system performance against a state of the art clinical FGS instrument, a direct sensitivity comparison with the Zeiss OPMT Pentero Blue 400 surgical microscope was conducted. Tissue-simulating liquid phantoms consisting of 1% intralipid, 1 mginl hemoglobin (Hemoglobin AO ferrous stabilized human, Sigma- Aldrich) and serial dilutions of PpIX were examined with both systems. Imaging was completed in ambient lighting conditions (— 125 μ\\½πι ) with the pulsed- light system but was performed in the dark following standard clinical practice using the Pentero operating microscope. The Pentero system excites PpIX with violet blue light (λ = 405 ± 5 nm), which coincides with the maximum absorption peak of the compound. Images from both systems as a function of PpIX concentration are presented in FIGS. 8A-8C. FIG. 8A: Zeiss Pentero surgical microscope (acquired in the dark) RGB images visible to the surgeon (RGB images are images containing red (R), green (G), and blue (B) color information), FIG. 8B: Pentero images from the red RGB channel only, and FIG. 8C: background subtracted images from the pulsed imaging system in ambient lighting. (Note: pulsed images smoothed using a median filter and color scale on all images chosen to show maximum contrast and no images are saturated.) Visible fluorescence images from the Pentero are shown as the surgeon would see them in FIG. 8A. FIG. 8B shows only red channel intensities extracted from the Pentero RGB images, and FIG. 8C presents the pulsed imaging system images of the same phantoms.

[0089] Contrast-to-noise ratios (CNR) 801 and raw signal 803 detected for the two systems are shown in FIGS. 8D and 8E, respectively, where red channel values from FIG. 8B were used to quantify the Pentero metrics. FIGS. 8D and 8E show fluorescence images of tissue-simulating phantoms containing different concentrations 802 of PpIX. FIGS. 8D and 8E show phantom resolution contrast to noise (calculated using central ROI in original images) and raw signal, respectively, for both systems where solid (810) and dashed (820) lines indicate signal in absence of PpIX. Results demonstrate the superior sensitivity to PpIX of the pulsed imaging system, which is observed despite detection under ambient light versus a dark room for the Pentero. The minimum visible concentration of PpIX was 0.25 uM for the pulsed FGS system and 5 μΜ for the Pentero (the Pentero did detect concentrations as low as 1 uM when examining the red channel only). Detected signal and contrast to noise were linear with concentration for both systems with coefficient of determination, R², values of 0.99 or greater.

[0090] The performance of the two systems during tumor resection of gliomas was also compared. Orthotopic U251 tumors were implanted in athymic nude mice 19 days prior to surgical imaging. To confirm the presence of tumors, gadolinium-enhanced MR images were acquired one day prior to surgery. For surgical imaging, mice were injected with 100 mg4cg of 5-aminolevulinic acid (ALA) i.p. 2 hours prior, which leads to accumulation of PpIX in tumor cells. Animals were anesthetized using isoflurane, placed in a stereotactic frame, and the skull cap removed to expose the brain. Mice were then imaged with the pulsed FGS system under ambient lights and the Pentero operating microscope in the dark.

[0091] FIGS. 9A-9H shows images from two separate mice with orthotopic U2 1 glioma. FIG. 9A: Preoperative MRJ Tl -weighted with gadolinium contrast (red arrow indicates tumor location). FIG. 9B: In vivo image of exposed brain using the Pentero. FIG. 9C: Background subtracted fluorescent in vivo image of exposed brain from the current system prior to any resection. FIG. 9D: Following resection and exposure of the tumor. FIG. 9E: Brain section stained with Hematoxylin (H) and eosin (E). FIG. 9F: Brain section stained with Hematoxylin and Eosin. FIG. 9G: Imaged with the Pentero. FIG. 9H, imaged with current pulsed-light system. (All pulsed-light fluorescent images overlaid on Pentero white light images use 60% thresholding and were obtained with room lights on.)

[0092] The mouse in FIGS. 9A-9D demonstrates video-rate imaging in vivo (Media 1) with the pulsed FGS system under ambient light conditions. The Tl -weighted gadolinium-enhanced MR image exhibiting tumor around 400 mm³ and below the surface is shown in FIG. 9A. No fluorescence enhancement was observed prior to resection with the Pentero system (FIG. 9B); however, the deeper penetration of red light from the pulsed FGS system revealed subsurface tumor tissue (FIG. 9C). As resection proceeds and tissue above the tumor is removed, a clear increase in fluorescence is evident as the tumor is exposed (FIG. 9D), demonstrating the highly sensitive, video rate, and subsurface detection potential of the pulsed imaging approach.

[0093] Other mice in the study were sacrificed following initial in vivo imaging, brains were extracted and sliced into four coronal sections, which were then imaged with both systems, processed for histology into formalin fixed paraffin embedded sections, and hematoxylin and eosin (H&E) stained. A representative case is shown in FIGS. 9E-9H. Ex vivo images in FIGS. 9G and 9H show the presence of a fluorescent subsurface mass with both systems, which was confirmed as tumor with the

corresponding H&E slide (FIGS. 9E and 9F).

[0094] These results suggest that pulsed fluorescence imaging can be deployed to facilitate FGS under ambient lighting conditions and without the need for specialized ambient-light filtering. The system described herein was shown to be sensitive to lower concentrations of ΡρΓΧ than the current state-of-the-art commercial fluorescence surgical microscope. Interpretation of sensitivity studies must take into account the difference in excitation wavelengths used by the two systems. The Pentero excites in the blue where PpIX excitation is far more efficient than in the red (30-fold higher molar extinction coefficient at 405 nm than at 630 nm). Additionally hemoglobin absorption at 405 nm is 600 fold greater than at 630 nm. Monte Carlo simulations showed that the remitted fluorescence intensity should two to three fold higher using 405 nm versus 630 nm excitation. Even with this disadvantage, the pulsed system had a far lower detection limit, which reinforces the value of pulsed imaging. Monte Carlo results also showed that 405 nm excitation produced substantially more surface-weighted measurements, with an average depth of fluorescence origin at 0.15 versus 1.02 mm for 630 nm excitation. These results further demonstrate the advantage of red light excitation for the detection of subsurface tumor sites.

[0095] Improved sensitivity during surgical resection of tumor is critical. In vivo visual detection of PpIX with the Pentero down to ~ 1.07 μΜ has been reported, while in vivo quantitative human data indicated a requirement for a detection threshold of 0.18 μΜ (roughly six times lower) to provide positive predictive values in excess of 90% and a detection threshold of ~0.02 uM (roughly 60 times lower) to achieve sensitivities in high-grade gliomas of >90%. These studies demonstrated that advanced optical detection techniques for PpIX fluorescence that achieve improved sensitivity will positively impact the diagnostic accuracy of FGS, suggesting the need for more sensitive technologies (advanced quantitative techniques, 87% accuracy versus visible techniques, 66%, p < 0.0001). The pulsed FGS system demonstrated detection to the level of 0.25 μΜ in realistic phantoms. Additionally, the UV Gen II sensor used here has a quantum efficiency (QE) of only 6% at 700 nm while other available sensors have QEs up to 35% at 700 nm, which should improve these results even further.

[0096] The use of the spectroscopy camera in these experiments not only resulted in lower resolution, but also in a lower frame rate, approximately seven frames per second (fps) in the absence of binning. Readout time from the ICCD is the primary limiting factor in regard to achieving the highest possible frame rates. The imaging version of the PI-MAX III has considerably faster readout times. The imaging version can be mounted on this system and is capable of achieving frame rates of 56 fps with pixels binned to match the 26 um pixel size of the spectroscopy camera and up to 140 fps when a 256 x 256 region of interest (ROI) is used.

[0097] The fast acquisition of pulsed-light imaging also holds promise for spectrally resolved FGS techniques. Current approaches, which require multiple images to construct a spectral image data cube, could be dramatically accelerated with this process. In recent years, significant advances in light filtered microscopes have occurred that allow NIR and narrowband optical imaging. The principles developed here for pulsed imaging are synergistic with these other filtering methods and could be combined to maximize signal to background ratios.

[0098] The system and concepts described in this example show considerable potential for enabling highly sensitive fluorescence imaging under normal room light conditions and also for detecting fluorescence at some depth in vivo. These advances may have considerable impact on improving efficacy in FGS as well as in moving the technology further into the clinic.

Example II: Pulsed-light imaging for fluorescence guided surgery with and without ambient light gating

[0099] The pulsed FGS system described in Example I relies on high-powered illumination to produce fluorescent signals that are detectible above the ambient light produced by normal fluorescent room lights. The present Example demonstrates the added benefit of gating fluorescence imaging on the ambient variation. This is equivalent to using FGS system 100 (FIG. 1) or 200 (FIG. 2) to perform one or more of methods 300 (FIG. 3), 400 (FIG. 4), and 600 (FIG. 6). It is shown that utilizing ambient light gating can not only further reduce ambient light contribution, as compared to results presented in Example I, but can also greatly reduce noise enabling fluorescent imaging under lighting conditions that would otherwise be impossible. This technique may prove essential for using the pulsed FGS system in an actual surgical operating room (OR) where overhead fluorescent lights are considerably stronger than standard room lighting. The present Example further examines the strength of different types of ambient light induced signals at the two channels, 700 nm and 800 nm, used for imaging with the pulsed system, as well as how image quality is affected by properties of the data acquisition.

[0100] The advantages provided by the use of high-powered pulsed light sources, as discussed in Example I, vary considerably depending upon the intensity and spectrum of ambient light. Ambient illumination within typical laboratory or surgical OR is generally provided by fluorescent white lights. The intensity and spectrum of these lights can vary considerably with the intensity found within a typical surgical operating room, which is considerably greater than that found in a standard lab setting. In the following, different lighting conditions are evaluated.

[0101] Typical laboratory lighting:

[0102] The ambient light in a typical laboratory will be examined here and its influence on the pulsed FGS system, described in Example I, at the 700 nm channel explored. A power meter was used to estimate average power to be 34.5 μ /cm .

Temporal measurements were also taken at a sample rate of 48kHz using a photodiode (DET10A Si Based Detector 200-1 100 nm, ThorLabs) and data acquisition (NI DAQ 6009) board with the resulting temporal signal displayed in FIG. 10A.

[0103] FIG. 10A shows temporal signal from standard laboratory and demonstrates a noisy, but relatively low signal which averages under 35 μ Υ/ϋηι² and varies by less than 3 μψ/cm². FIGS. 10B and IOC show spectrum recorded in the same standard laboratory on a linear scale and a log scale, respectively. Signal at 700nm is substantially larger than that at 800 nm. Laboratory used a series of Sylvania Octron XP 17W 3500K overhead fluorescent lights. The spectrum from the overhead room lights was recorded with a spectrometer (Ocean Optics, QE 65000) and is provided in FIGS. 10B and IOC. It can be seen that there is substantially greater signal in the vicinity of 700 nm than in the vicinity of 800 nm. Readings taken using a power meter (ThorLabs) where the sensor was covered by a 700 nm 40BP filter (Omega) and then an 800 nm 40BP filter

2 2

(Omega) produced measurements of 5.0 uW/cm and 0.4 uW/cm respectively within the area of imaging. This amounts to an approximately 12-fold greater background signal at the 700 nm channel than at the 800 nm channel. Given these results, it would be expected that the use of short integration times and pulsed light would provide greater benefits when looking at lower wavelength (i.e. 700 nm) fluorophores.

[0104] A set of tissue simulating phantoms composed of water, 1% intralipid and 0.024% india ink (absorption coefficient Ua = 0.05 cm^"1) were constructed with serial dilutions of IRDye 680RD. These phantoms were examined under a number of different camera and LED settings in order to evaluate the detection limit capabilities under different configurations.

[0105] FIG. 11 shows a selection of IRDye 680RD phantom images, captured in a standard laboratory, under different acquisition settings. The lowest detection levels and best contrast to noise ratios (CNR) were found using either short, sub millisecond gate widths, full gain and LED overdriving or at substantially longer exposure times of 100ms or greater. These results suggest that while imaging using the 700 nm channel in normal lab room light is feasible without pulsing or intensifier gain, the time required to obtain comparable images is substantially longer and will preclude video rate imaging. As these images are background subtracted, a gate width of 100 ms in fact requires 200 ms to acquire both images and so has a maximum frame rate of 5 frames per second (fps). [0106] The series of phantoms discussed in connection with FIG. 1 1 show that imaging at the 700 nm channel in normal room light is feasible without using short gate widths to suppress ambient light induced background.

[0107] Tungsten halogen surgical lamp lighting:

[0108] The tungsten halogen surgical lamp is currently the standard in the operating room although there is some movement toward the use of LED surgical lamps. Surgical lamps produce high powered, uniform illumination with minimal shadowing providing the surgeon with a clear, bright and consistent view of the surgical cavity. The spectrum and temporal signal of the Sylvania 150P25/2SB clear silver bowl spot lamp was examined and the results are plotted in FIGS. 12A-12C. FIGS. 12A-12C show temporal signal from tungsten halogen incandescent surgical lamp and demonstrates 120 Hz output. FIGS. 12B and 12C show spectrum from the same surgical lamp displayed on a linear scale and log scale, respectively. Output from the lamp is periodic at 120 Hz as shown in FIG. 12A, but amplitude variations are relatively small in comparison to average power (less than 10% variation about the mean). The spectrum, plotted in FIGS. 12B and 12C, shows considerable power in the 700 nm and 800 nm ranges as is to be expected from a tungsten halogen lamp.

[0109] The same series of IRDye 680RD phantoms described in Example I were imaged with the tungsten halogen surgical light directly illuminating them. Power measurements (details described in previous section) showed 290

at the 700 nm channel with the surgical light set to half power. Several different camera settings were tested to get an idea of what the optimum settings might be under this type of extremely high ambient light brightness condition. A series of images were taken at gate widths of 10 is, 20 is and 40 with the intensifier gain set to 100% as well as at 1ms with no gain.

[0110] FIGS. 13 A and 13B illustrate minimal detectible concentrations under high intensity surgical lighting using a tungsten halogen surgical lamp. FIG. 13 A shows a box and whisker plot from the images taken at 40 and displays the variation in signal across thirty fluorescence images (upper boxes in each column) and thirty background images (lower boxes in each column). The central lines are the medians while the edges of the boxes are the 25th and 75th percentiles. Significant variation between images can be seen and contributes to the higher detection limits that were observed as compared to under normal fluorescent ambient light. Detection limits here are defined as the lowest concentration where the box and whiskers from the fluorescent images and the background images do not overlap. FIG. 13B shows minimum detectible signals for different camera settings, where it can be seen that further reduction in gate width resulted in decreased performance, as did the elimination of gain and an increase in gate width to 1 ms. Figure 13B shows that neither a reduction in gate width and continued use of gain, nor an increase in gate width to 1ms and a disabling of gain, improved limits of detection.

[0111] High intensity fluorescent surgical lighting:

[0112] Analyses of signal from fluorescent overhead lights in two actual surgical ORs indicate considerably higher power than is seen in a typical office or lab environment as well as a substantial periodic signal fluctuation. A power meter (PM100, ThorLabs,) was used to estimate average power in the two rooms to be 182 and 124 μλν/cm². Temporal measurements were also taken at a sample rate of 48 kHz using a photodiode with both surgical rooms displaying strong periodic fluctuations at 120 Hz or double the electric power frequency. Periodic signal from the two surgical rooms is plotted in FIG 14A, clearly showing the periodic nature of the signal and the 120 Hz frequency. Spectral measurements (QE 65000, Ocean Optics) were also taken and those are plotted in FIGS. 14B and 14C, on a linear and log scale, respectively, where it is seen that substantially greater signal is present in the 700 nm range than in the 800 nm range as expected.

[0113] The Pulsed FGS system, described in Example I, was configured to allow triggering directly from an input signal provided by the ambient lights. An operational amplifier (Op Amp) (Texas Instruments, LM741) was used to amplify the voltage provided by a photodiode (DET10A Si Based Detector 200-1100 nm, ThorLabs) which was positioned to monitor ambient lights. The amplified analog signal was then sampled via a DAQ board (NI USB-6351, X Series DAQ). The NI-6351 is capable of sending multiple digital triggers based on an analog input and this can be done from entirely within the hardware. The signal level used to trigger image acquisition was adjusted to time image acquisition to the period of minimal ambient light brightness.

[0114] Ambient light based triggering at the 700 nm channel under high- intensity surgical lighting:

[0115] Fluorescence detection levels in the surgical OR (surgical room 1) were tested using liquid tissue simulating phantoms. Serial dilutions of IRDye 680RD (1% intralipid and 0.01% India ink for absorption coefficient μ_& = 0.02 cm^"1 and reduced scattering coefficient μ₅' = 24 cm^"1) were examined under a variety of acquisition settings. FIGS. 15A and 15B shows results obtained using the Pulsed FGS system, described in Example I, without and with room light based triggering as discussed in connection with FIGS. 1 through 4. FIGS. 15A and 15B show box and whisker plot of ambient light subtracted signal for series of 50 images taken at each IRDye 680RD concentration from 0 to 3.9 nM. This is for 1ms gate widths, full camera gain and lOx overdriving of the LEDs. FIG. 15 A shows results based upon images taken in the surgical OR with no room light based triggering. FIG. 15B shows results based upon images taken in the surgical OR with room light based triggering (offset left) and images taken in complete darkness (offset right). For all box and whisker plots the central line is the median, box edges are the 25th and 75th percentile. This can be seen in FIG. 1 B where the same acquisition settings are used but this time triggering is either based on room light signal (offset left) or imaging is done in complete darkness (offset right). The reduction in signal variation is drastic and the ambient light subtracted signal using ambient light based triggering is nearly identical to that seen when imaging in a completely darkened room.

[0116] The results shown in FIG. 15B with room light based triggering utilizes the ambient light subtraction method discussed in connection with FIG. 6. The signal level in the original images captured under ambient light are considerably higher than those seen in the darkened room. It is only after ambient light subtraction that the two become comparable.

[0117] Ambient light based triggering at the 800 nm channel under high- intensity surgical lighting:

[0118] Fluorescence detection levels in the surgical OR (surgical room 1) at the 800 nm channel were tested in the same manner as those described in the previous section for the 700 nm channel. Serial dilutions of IRDye 800RD were examined under a variety of acquisition settings.

[0119] Despite reduced ambient light contribution at the 800 nm channel as compared to the 700 nm channel the same problem created by the large 120 Hz fluctuations make standard sub-millisecond pulsed imaging impractical at lower fluorophore concentrations. As illustrated in FIGS. 16A and 16B ambient light gating is necessary for fluorescence imaging. FIGS. 16A and 16B shows results obtained using the Pulsed FGS system, described in Example I, without and with room light based triggering as discussed in connection with FIGS. 1 through 4. FIGS. 16A and 16B show box and whisker plot of ambient light subtracted signal for series of 50 images taken at each IRDye 800CW concentration from 0 to 3.9 nM. This is for 1ms gate widths, full camera gain and l Ox overdriving of the LEDs. FIG. 16A shows results based upon images taken in the surgical OR with no room light based triggering. FIG. 16B shows results based upon images taken in the surgical OR with room light based triggering (offset left) and images taken in complete darkness (offset right). For all box and whisker plots the central line is the median, box edges are the 25th and 75th percentile. This can be seen in FIG. 16B where the same acquisition settings are used but this time triggering is either based on room light signal (offset left) or imaging is done in complete darkness (offset right). The reduction in signal variation is drastic and the ambient light subtracted signal using ambient light based triggering is nearly identical to that seen when imaging in a completely darkened room.

Example III: Simulated Pulsed-light imaging for fluorescence guided surgery with ambient light gating

[0120] Example III demonstrates the benefit of gating fluorescence imaging on periods of identical, or substantially identical periods of ambient light brightness. In the Example, the periods are periods of low ambient light brightness. However, significant benefit may be achieved also when using periods of greater, substantially identical ambient light brightness.

[0121] FIG. 17A shows time-domain ambient light variation for two surgical rooms lit by high intensity fluorescent lights. The ambient light power in both surgical rooms is significant and displays a frequency of 120 Hz, or double the utility frequency. The light power of surgical room 1 has a DC component of 123

with an amplitude of 51 μ Λ;πι², and that of surgical room 2 has a DC component of 223

[0122] FIG. 17B shows simulated fluorescence and ambient light data. The data is based on actual measurements acquired with a pulsed FGS system, such as FGS system 200 (FIG. 2) without gating on ambient light, using 500 s gate widths and the 800 nm channel in surgical room 2 as well as fluorescence measurements made in tissue simulating phantoms (1% intralipid, absorption coefficient = 0.05 cm-1, and 5 nM IRDye 800). Figure 17B demonstrates the problem of achieving accurate ambient light subtraction when not gating on the ambient light brightness

[0123] FIG. 17C shows simulated fluorescence and ambient light data using acquisition rate of 30 Hz to down sample room light frequency and demonstrate technique to enable extraction of fluorescence signal. Thus, FIG. 17C simulates results that may be obtained using FGS system 200 (FIG. 2) together with methods 400 and 600 of FIGS. 4 and 6, respectively. As evident in FIG. 17C, the fluorescent signal is discernible due to the implementation of fluorescence imaging synchronized with periods of minimal ambient light brightness and use of ambient light subtraction as discussed in FIG. 6.

[0124] Combinations of features

[0125] Features described above as well as those claimed below may be combined in various ways without departing from the scope hereof. For example, it will be appreciated that aspects of one fluorescence guided surgical system, or method, described herein may incorporate or swap features of another fluorescence guided surgical system, or method, described herein. The following examples illustrate possible, non- limiting combinations of embodiments described above. It should be clear that many other changes and modifications may be made to the systems and methods described herein without departing from the spirit and scope of this invention:

[0126] (Al) A fluorescence guided surgical system gated on ambient light, may include a fluorescence imaging system for imaging a fluorescing agent in a subject during surgery on the subject, and a sensor for detecting periods of low brightness of the ambient light to activate the imaging only during the periods of low brightness.

[0127] (A2) In the fluorescence guided surgical system denoted (Al), the sensor may be an ambient light sensor for sensing brightness of the ambient light.

[0128] (A3) In the fluorescence guided surgical system denoted as (Al), the sensor may be configured to sense oscillations of alternating current driving an ambient light source generating the ambient light.

[0129] (A4) In each of the fluorescence guided surgical systems denoted as (A 1) through (A3), the periods of low brightness may be periods of minimal brightness.

[0130] (A5) In each of the fluorescence guided surgical systems denoted as (A 1) through (A4), the periods of low brightness may be periods of substantially identical brightness. [0131] (A6) In each of the fluorescence guided surgical systems denoted as (Al) through (A5), the fluorescence imaging system may be configured for capturing images of fluorescence in a wavelength range that includes (a) at least a portion of the visible spectrum and (b) some wavelengths of the ambient light.

[0132] (A7) Each of the fluorescence guided surgical systems denoted as (Al) through (A6) may further include a control module for controlling on and off states of the fluorescence imaging system according to input from the sensor.

[0133] (A8) The fluorescence guided surgical system denoted as (A7) may further include an illumination module for emitting excitation light to excite the fluorescing agent.

[0134] (A9) In the fluorescence guided surgical system denoted as (A8), the illumination module may include an interface for receiving, from the control module, a control signal causing emission of a plurality of light pulses, wherein each of the plurality of light pulses is emitted during a respective one of the periods of low brightness of the ambient light.

[0135] (A 10) In the fluorescence guided surgical system denoted as (A9), the plurality of light pulses may have duration and power such that (a) time-averaged power, delivered to the subject and averaged over the plurality of the light pulses, is below a safety-related upper limit, and (b) energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of the fluorescence imaging system.

[0136] (Al 1) In the fluorescence guided surgical system denoted as (A 10), the fluorescence imaging system may include an illumination module for inducing fluorescence of the fluorescing agent, and a camera for imaging the fluorescence induced by the illumination module.

[0137] (A 12) In the fluorescence guided surgical system denoted as (Al l), the illumination module may include a light source for emitting excitation light to induce the fluorescence, and a focusing element for focusing the excitation light onto an area of interest of the subject.

[0138] (A13) In the fluorescence guided surgical system denoted as (A12), the light source may include at least one light-emitting diode.

[0139] (A14) In the fluorescence guided surgical system denoted as (A13), the at least one light-emitting diode may be a plurality of light-emitting diodes. [0140] (Al 5) In each of the fluorescence guided surgical systems denoted as (A 12) through (A 14), the focusing element may include a Fresnel lens.

[0141] (A 16) In each of the fluorescence guided surgical systems denoted as (Al 1) through (A15), the camera may include at least one wavelength filter, wherein each of the at least one wavelength filter transmits at least a portion of the fluorescence and blocks at least a portion of light different from the at least a portion of the fluorescence.

[0142] (A 17) In the fluorescence guided surgical system denoted as (A 16), the at least one wavelength filter may include a wavelength filter for transmitting light in a wavelength range that includes wavelength of at least a portion of the ambient light.

[0143] (Al 8) In each of the fluorescence guided surgical systems denoted as (A 16) through (A 17), the fluorescing agent comprising a plurality of different fluorescing agents having a respective plurality of different fluorescence wavelength ranges, wherein the at least one wavelength filter includes a plurality of wavelength filters for transmitting fluorescence associated with the respective plurality of different fluorescing agents.

[0144] (A 19) In each of the fluorescence guided surgical systems denoted as (Al l) through (Al 8), the camera may include a charge-coupled device image sensor.

[0145] (A20) In the fluorescence guided surgical system denoted as (A 19), the charge-coupled device image sensor may include an intensified charge-coupled device image sensor.

[0146] (A21) In the fluorescence guided surgical system denoted as (A 19), the charge-coupled device image sensor may include an electron-multiplying charge-coupled device image sensor.

[0147] (A22) In each of the fluorescence guided surgical systems denoted as (Al 1) through (A21), the camera may include an interface for receiving, from the control module, a control signal causing capture of a plurality of fluorescence images during respective ones of the periods of low brightness of the ambient light.

[0148] (A23) Each of the fluorescence guided surgical systems denoted as (Al l) through (A22) may further include an analysis module for receiving a pair of fluorescence images captured by the camera during at least a portion of the periods of low brightness of the ambient light, wherein the pair of fluorescence images includes (a) a signal fluorescence image captured while the illumination module is on and (b) a background fluorescence image captured while the illumination module is off, and wherein the analysis module includes instructions for generating an ambient light subtracted fluorescence image from the pair of fluorescence images.

[0149] (B 1 ) An ambient light gated method for generating fluorescence images to guide a surgical procedure may include detecting periods of low brightness of ambient light, and imaging a fluorescing agent in a subject undergoing surgery during a first subset of the periods of low brightness of the ambient light.

[0150] (B2) In the ambient light gated method denoted as (B 1), the step of detecting may include sensing brightness of the ambient light.

[0151] (B3) In the ambient light gated method denoted as (Bl), the step of detecting may include sensing oscillations of alternating current driving the ambient light.

[0152] (B4) In each of the ambient light gated methods denoted as (Bl) through (B3), the step of detecting may include detecting periods of minimal brightness of the ambient light.

[0153] (B5) In each of the ambient light gated methods denoted as (Bl) through (B4), the step of imaging may include inducing fluorescence of the fluorescing agent with fluorescence excitation light, and capturing fluorescence images of the subject during the first subset of the periods of low brightness of the ambient light.

[0154] (B6) The ambient light gated method denoted as (B5) may further include communicating a first control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of capturing in coincidence with the first subset of the periods of low brightness of the ambient light.

[0155] (B7) The ambient light gated method denoted as (B6) may further include communicating a second control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of inducing in coincidence with a second subset of the periods of low brightness of the ambient light.

[0156] (B8) In the ambient light gated method denoted as (B7), the second subset may be associated with a subset of the periods of low brightness of the ambient light that are associated with the first subset such that the step of capturing produces (a) at least one signal fluorescence image captured while performing the step of inducing and (b) at least one background fluorescence image captured while not performing the step of inducing. [0157] (B9) The ambient light gated method denoted as (B8) may further include generating an ambient light subtracted fluorescence image by subtracting one of the at least one background fluorescence image from a corresponding one of the at least one signal fluorescence image.

[0158] (B 10) In each of the ambient light gated methods denoted as (B5) through (B9), the step of inducing may include exposing the subject to a plurality of fluorescence excitation light pulses during a second respective subset of the periods of low brightness of the ambient light.

[0159] (B 1 1 ) In the ambient light gated method denoted as (B 10), the step of inducing may include exposing the subject to a plurality of fluorescence excitation light pulses having duration and power such that time-averaged power, delivered to the subject and averaged over the plurality of the fluorescence excitation light pulses, is below a safety-related upper limit.

[0160] (B 12) In the ambient light gated method denoted as (B 11 ), the plurality of fluorescence excitation light pulses may further have duration and power such that energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of a camera.

[0161] (B 13) In each of the ambient light gated methods denoted as (B5) through (B12), the step of inducing may include focusing the fluorescence excitation light onto an area of interest of the subject.

[0162] (B14) In the ambient light gated method denoted as (B13), the step of inducing may further include generating the fluorescence excitation light using at least one light-emitting diode.

[0163] (B15) In each of the ambient light gated methods denoted as (B5) through (B14), the step of capturing may include filtering light, using at least one wavelength filter, to reduce contribution to the fluorescence images from light different from the fluorescence.

[0164] (B 16) In each of the ambient light gated methods denoted as (B5) through (B15), the step of inducing may include exciting, in the subject, a plurality of different fluorescing agents, having a respective plurality of different fluorescence wavelength ranges; and the step of capturing may include capturing a respective plurality of fluorescence image sets while filtering light, using a respective plurality of wavelength filters, to reduce contribution to the plurality of fluorescence image sets from light not associated with the respective plurality of different fluorescing agents.

[0165] (B 17) In each of the ambient light gated methods denoted as (B5) through (B16), the step of capturing may include capturing the fluorescence images using an image sensor selected from the group of an intensified charge-coupled device image sensor and an electron-multiplied charge-coupled device image sensor.

[0166] (B 18) Each of the ambient light gated methods denoted as (B 1 ) through (B 17) may further include generating the ambient light as fluorescent light exhibiting periodic brightness variation.

[0167] (CI) A fluorescence guided surgical system gated on ambient light may include a fluorescence detection system for detecting fluorescence from a fluorescing agent in a subject during surgery thereon, and a sensor for detecting periods of low brightness of the ambient light to activate the fluorescence detection only during the periods of low brightness.

[0168] (C2) In the fluorescence guided surgical system denoted as (CI), the sensor may be an ambient light sensor for sensing brightness of the ambient light.

[0169] (C3) In the fluorescence guided surgical system denoted as (CI), the sensor may be configured to sense oscillations of alternating current driving an ambient light source generating the ambient light.

[0170] (C4) Each of the fluorescence guided surgical systems denoted as (C 1) through (C3) may further include a control module for controlling on and off states of the fluorescence detection system according to input from the sensor.

[0171] (C5) In the fluorescence guided surgical system denoted as (C4), the fluorescence detection system may include an illumination module for emitting excitation light to excite the fluorescing agent.

[0172] (C6) In the fluorescence guided surgical system denoted as (C5), the illumination module may include an interface for receiving, from the control module, a control signal causing emission of a plurality of light pulses, during respective ones of the periods of low brightness of the ambient light.

[0173] (C7) In the fluorescence guided surgical system denoted as (C6), the plurality of light pulses may have duration and power such that (a) time-averaged power, delivered to the subject and averaged over the plurality of the light pulses, is below a safety-related upper limit, and (b) energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of the fluorescence detection system.

[0174] (C8) In each of the fluorescence guided surgical systems denoted as (C 1 ) through (C7), the fluorescence detection system may include an illumination module for inducing fluorescence, and a detector for detecting the fluorescence induced by the illumination module and generating a value indicative of the fluorescence detected.

[0175] (C9) The fluorescence guided surgical system denoted as (C8) may further include an analysis module for receiving a pair of values generated by the detector during at least a portion of the periods of low brightness of the ambient light, wherein the pair of values includes (a) a signal value generated while the illumination module is on and (b) a background value generated while the illumination module is off, and wherein the analysis module includes instructions for generating an ambient light subtracted fluorescence value from the pair of values.

[0176] (Dl) An ambient light gated method for generating fluorescence images to guide a surgical procedure may include (a) detecting periods of low brightness of ambient light, (b) inducing fluorescence in a subject undergoing surgery with fluorescence excitation light, and (c) measuring fluorescence of the subject during a first subset of the periods of low brightness of the ambient light.

[0177] (D2) In the ambient light gated method denoted as (Dl), the step of detecting may include sensing brightness of the ambient light.

[0178] (D3) In the ambient light gated method of denoted as (Dl), the step of detecting may include sensing oscillations of alternating current driving the ambient light.

[0179] (D4) Each of the ambient light gated methods denoted as (Dl ) through (D3) may further include communicating a first control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of measuring in coincidence with the first subset of the periods of low brightness of the ambient light.

[0180] (D5) The ambient light gated method denoted as (D4) may further include communicating a second control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting to trigger performance of the step of inducing in coincidence with a second subset of the periods of low brightness of the ambient light. [0181] (D6) In the ambient light gated method denoted as (D5), the second subset may be associated with a subset of the periods of low brightness of the ambient light that are associated with the first subset such that the step of measuring produces (a) at least one signal value generated while performing the step of inducing and (b) at least one background value generated while not performing the step of inducing.

[0182] (D7) The ambient light gated method denoted as (D6) may further include generating an ambient light subtracted fluorescence value by subtracting one of the at least one background value from a corresponding one of the at least one signal value.

[0183] (D8) In each of the ambient light gated methods denoted as (Dl) through (D7), the step of inducing may include exposing the subject to a plurality of fluorescence excitation light pulses during a second respective subset of the periods of low brightness of the ambient light.

[0184] (D9) In the ambient light gated method denoted as (D8), the step of inducing may include exposing the subject to a plurality of fluorescence excitation light pulses having duration and power such that time-averaged power, delivered to the subject and averaged over the plurality of the fluorescence excitation light pulses, is below a safety-related upper limit.

[0185] (D10) In the ambient light gated method denoted as (D9), the plurality of fluorescence excitation light pulses may further have duration and power such that energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of a detector.

[0186] Changes may be made in the above systems and methods without departing from the scope hereof. It should thus be noted that the matter contained in the above description and shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover generic and specific features described herein, as well as all statements of the scope of the present method and device, which, as a matter of language, might be said to fall therebetween.

Claims

What is claimed is:

1. A fluorescence guided surgical system gated on ambient light, comprising: fluorescence imaging system for imaging a fluorescing agent in a subject during surgery on the subject; and

sensor for detecting periods of low brightness of the ambient light to activate the imaging only during the periods of low brightness.

2. The fluorescence guided surgical system of claim 1, the sensor being an ambient light sensor for sensing brightness of the ambient light.

3. The fluorescence guided surgical system of claim 1, the sensor being configured to sense oscillations of alternating current driving an ambient light source generating the ambient light.

4. The fluorescence guided surgical system of claim 1 , the periods of low brightness being periods of minimal brightness.

5. The fluorescence guided surgical system of claim 1, the periods of low brightness being periods of substantially identical brightness.

6. The fluorescence guided surgical system of claim 1, the fluorescence imaging system configured for capturing images of fluorescence in a wavelength range that includes (a) at least a portion of the visible spectrum and (b) some wavelengths of the ambient light.

7. The fluorescence guided surgical system of claim 1, further comprising a control module for controlling on and off states of the fluorescence imaging system according to input from the sensor.

8. The fluorescence guided surgical system of claim 7, the fluorescence imaging system comprising an illumination module for emitting excitation light to excite the fluorescing agent, the illumination module comprising an interface for receiving, from the control module, a control signal causing emission of a plurality of light pulses, each of the plurality of light pulses being emitted during a respective one of the periods of low brightness of the ambient light.

9. The fluorescence guided surgical system of claim 8, the plurality of light pulses having duration and power such that (a) time-averaged power, delivered to the subject and averaged over the plurality of the light pulses, is below a safety-related upper limit, and (b) energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of the fluorescence imaging system.

10. The fluorescence guided surgical system of claim 1, the fluorescence imaging system comprising:

illumination module for inducing fluorescence of the fluorescing agent; and camera for imaging the fluorescence induced by the illumination module.

1 1. The fluorescence guided surgical system of claim 10, the illumination module comprising:

light source for emitting excitation light to induce the fluorescence; and focusing element for focusing the excitation light onto an area of interest of the subject.

12. The fluorescence guided surgical system of claim 11, the light source comprising at least one light-emitting diode.

13. The fluorescence guided surgical system of claim 12, the at least one light- emitting diode being a plurality of light-emitting diodes.

14. The fluorescence guided surgical system of claim 11, the focusing element comprising a Fresnel lens.

15. The fluorescence guided surgical system of claim 10, the camera comprising at least one wavelength filter, each of the at least one wavelength filter transmitting at least a portion of the fluorescence and blocking at least a portion of light different from the at least a portion of the fluorescence.

16. The fluorescence guided surgical system of claim 15, the at least one wavelength filter comprising a wavelength filter for transmitting light in a wavelength range that includes wavelength of at least a portion of the ambient light.

17. The fluorescence guided surgical system of claim 15, the fluorescing agent comprising a plurality of different fluorescing agents having a respective plurality of different fluorescence wavelength ranges, the at least one wavelength filter comprising a plurality of wavelength filters for transmitting fluorescence associated with the respective plurality of different fluorescing agents.

18. The fluorescence guided surgical system of claim 10, the camera comprising a charge-coupled device image sensor.

19. The fluorescence guided surgical system of claim 18, the charge-coupled device image sensor comprising an intensified charge-coupled device image sensor.

20. The fluorescence guided surgical system of claim 18, the charge-coupled device image sensor comprising an electron-multiplying charge-coupled device image sensor.

21. The fluorescence guided surgical system of claim 10, the camera comprising an interface for receiving, from the control module, a control signal causing capture of a plurality of fluorescence images during respective ones of the periods of low brightness of the ambient light.

22. The fluorescence guided surgical system of claim 10, further comprising an analysis module for receiving a pair of fluorescence images captured by the camera during at least a portion of the periods of low brightness of the ambient light, the pair of fluorescence images including (a) a signal fluorescence image captured while the illumination module is on and (b) a background fluorescence image captured while the illumination module is off, the analysis module including instructions for generating an ambient light subtracted fluorescence image from the pair of fluorescence images.

23. An ambient light gated method for generating fluorescence images to guide a surgical procedure, comprising:

detecting periods of low brightness of ambient light; imaging a fluorescing agent in a subject undergoing surgery during a first subset of the periods of low brightness of the ambient light.

24. The ambient light gated method of claim 23, the step of detecting comprising sensing brightness of the ambient light.

25. The ambient light gated method of claim 23, the step of detecting comprising sensing oscillations of alternating current driving the ambient light.

26. The ambient light gated method of claim 23, the step of detecting comprising detecting periods of minimal brightness of the ambient light.

27. The ambient light gated method of claim 23, the step of imaging comprising:

inducing fluorescence of the fluorescing agent with fluorescence excitation light; and

capturing fluorescence images of the subject during the first subset of the periods of low brightness of the ambient light.

28. The ambient light gated method of claim 27, further comprising communicating a first control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of capturing in coincidence with the first subset of the periods of low brightness of the ambient light.

29. The ambient light gated method of claim 28, further comprising communicating a second control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of inducing in coincidence with a second subset of the periods of low brightness of the ambient light.

30. The ambient light gated method of claim 29, the second subset being associated with a subset of the periods of low brightness of the ambient light that are associated with the first subset such that the step of capturing produces (a) at least one signal fluorescence image captured while performing the step of inducing and (b) at least one background fluorescence image captured while not performing the step of inducing.

31. The ambient light gated method of claim 30, further comprising generating an ambient light subtracted fluorescence image by subtracting one of the at least one background fluorescence image from a corresponding one of the at least one signal fluorescence image.

32. The ambient light gated method of claim 27, the step of inducing comprising exposing the subject to a plurality of fluorescence excitation light pulses during a second respective subset of the periods of low brightness of the ambient light.

33. The ambient light gated method of claim 32, the step of inducing comprising exposing the subject to a plurality of fluorescence excitation light pulses having duration and power such that time-averaged power, delivered to the subject and averaged over the plurality of the fluorescence excitation light pulses, is below a safety- related upper limit.

34. The ambient light gated method of claim 33, the plurality of fluorescence excitation light pulses further having duration and power such that energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of a camera.

35. The ambient light gated method of claim 27, the step of inducing comprising focusing the fluorescence excitation light onto an area of interest of the subject.

36. The ambient light gated method of claim 35, the step of inducing further comprising generating the fluorescence excitation light using at least one light-emitting diode.

37. The ambient light gated method of claim 27, the step of capturing comprising filtering light, using at least one wavelength filter, to reduce contribution to the fluorescence images from light different from the fluorescence.

38. The ambient light gated method of claim 23, further comprising generating the ambient light as fluorescent light exhibiting periodic brightness variation.

39. The ambient light gated method of claim 27, the step of inducing comprising exciting, in the subject, a plurality of different fluorescing agents, having a respective plurality of different fluorescence wavelength ranges; and

the step of capturing comprising capturing a respective plurality of fluorescence image sets while filtering light, using a respective plurality of wavelength filters, to reduce contribution to the plurality of fluorescence image sets from light not associated with the respective plurality of different fluorescing agents.

40. The ambient light gated method of claim 27, the step of capturing comprising capturing the fluorescence images using an image sensor selected from the group of an intensified charge-coupled device image sensor and an electron-multiplied charge-coupled device image sensor.

41. A fluorescence guided surgical system gated on ambient light, comprising: fluorescence detection system for detecting fluorescence from a fluorescing agent in a subject during surgery thereon; and

sensor for detecting periods of low brightness of the ambient light to activate said detection only during the periods of low brightness.

42. The fluorescence guided surgical system of claim 41, the sensor being an ambient light sensor for sensing brightness of the ambient light.

43. The fluorescence guided surgical system of claim 41, the sensor being configured to sense oscillations of alternating current driving an ambient light source generating the ambient light.

44. The fluorescence guided surgical system of claim 41, further comprising a control module for controlling on and off states of the fluorescence detection system according to input from the sensor.

45. The fluorescence guided surgical system of claim 44, the fluorescence detection system comprising an illumination module for emitting excitation light to excite the fluorescing agent, the illumination module comprising an interface for receiving, from the control module, a control signal causing emission of a plurality of light pulses, during respective ones of the periods of low brightness of the ambient light.

46. The fluorescence guided surgical system of claim 45, the plurality of light pulses having duration and power such that (a) time-averaged power, delivered to the subject and averaged over the plurality of the light pulses, is below a safety-related upper limit, and (b) energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of the fluorescence detection system.

47. The fluorescence guided surgical system of claim 41, the fluorescence detection system comprising:

an illumination module for inducing fluorescence; and

a detector for detecting the fluorescence induced by the illumination module and generating a value indicative of the fluorescence detected.

48. The fluorescence guided surgical system of claim 47, further comprising an analysis module for receiving a pair of values generated by the detector during at least a portion of the periods of low brightness of the ambient light, the pair of values including (a) a signal value generated while the illumination module is on and (b) a background value generated while the illumination module is off, the analysis module including instructions for generating an ambient light subtracted fluorescence value from the pair of values.

49. An ambient light gated method for generating fluorescence images to guide a surgical procedure, comprising:

detecting periods of low brightness of ambient light;

inducing fluorescence in a subject undergoing surgery with fluorescence

excitation light; and

measuring fluorescence of the subject during a first subset of the periods of low brightness of the ambient light.

50. The ambient light gated method of claim 49, the step of detecting comprising sensing brightness of the ambient light.

51. The ambient light gated method of claim 49, the step of detecting comprising sensing oscillations of alternating current driving the ambient light.

52. The ambient light gated method of claim 49, further comprising communicating a first control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting, to trigger performance of the step of measuring in coincidence with the first subset of the periods of low brightness of the ambient light.

52. The ambient light gated method of claim 52, further comprising communicating a second control signal, based upon the periods of low brightness of the ambient light detected in the step of detecting to trigger performance of the step of inducing in coincidence with a second subset of the periods of low brightness of the ambient light.

54. The ambient light gated method of claim 52, the second subset being associated with a subset of the periods of low brightness of the ambient light that are associated with the first subset such that the step of measuring produces (a) at least one signal value generated while performing the step of inducing and (b) at least one background value generated while not performing the step of inducing.

55. The ambient light gated method of claim 54, further comprising generating an ambient light subtracted fluorescence value by subtracting one of the at least one background value from a corresponding one of the at least one signal value.

56. The ambient light gated method of claim 49, the step of inducing comprising exposing the subject to a plurality of fluorescence excitation light pulses during a second respective subset of the periods of low brightness of the ambient light.

57. The ambient light gated method of claim 56, the step of inducing comprising exposing the subject to a plurality of fluorescence excitation light pulses having duration and power such that time-averaged power, delivered to the subject and averaged over the plurality of the fluorescence excitation light pulses, is below a safety- related upper limit.

58. The ambient light gated method of claim 57, the plurality of fluorescence excitation light pulses further having duration and power such that energy of each of at least a portion of the plurality of light pulses is sufficient to induce fluorescence above fluorescence-detection limit of a detector.