Patent Publication Number: US-2021166806-A1

Title: Methods and systems for adaptive imaging for low light signal enhancement in medical visualization

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 15/623,100, filed Jun. 14, 2017, which claims the benefit of U.S. Provisional Application No. 62/350,121, filed Jun. 14, 2016, the entire contents of each of which are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present disclosure relates generally to medical imaging. More specifically, the disclosure relates to adaptive imaging for low light signal enhancement in medical visualization. 
     BACKGROUND OF THE INVENTION 
     Imaging technology used in medical visualization (e.g., invasive, minimally-invasive, or non-invasive visualization) can suffer performance degradation under low light conditions. In particular, fluorescence imaging systems used for medical visualization may need to operate with very low emitted signal levels from fluorophores at low concentration, with limited quantum efficiency and/or deeply embedded in the tissue. 
     Low signal levels are not only problematic in cases of fluorescence imaging, however, but are also known to limit the signal quality of reflected light laparoscopic images which are acquired through small aperture optics (e.g. laparoscopes). The combination of low light signal sources (e.g. fluorescence) and small aperture optics (e.g. laparoscopes) may compound the challenge. 
     Medical imaging systems (e.g., endoscopic imaging systems for minimally-invasive surgery or open field medical imaging systems) can help provide clinical information for medical practitioners who need to make decisions (e.g. intraoperative or treatment decisions) based on visualization of tissue. In many applications, it is useful for medical imaging systems to provide white light video in combination with another imaging modality (e.g., fluorescence video) substantially simultaneously and in real-time. In particular, in applications for visualizing tissue, the white light video is typically acquired by illuminating the tissue with full visible spectrum light and imaging the illumination light that is reflected from the tissue surface. In typical applications, such white light video ideally maintains a high color fidelity with the image that would be perceived with the normal human eye directly visualizing the same reflected light. Additionally, fluorescence video, for example, may be acquired by illuminating the tissue with excitation light and imaging the fluorescence light that is emitted by excited fluorophores located in the tissue. The white light video (e.g., color video) and the fluorescence video may be merged and presented to the medical practitioner as a single “real-time” video. 
     There are occasions, however, when there may be a significant disparity between the intensity levels of the reflected illumination light and the fluorescence light. In particular, in many instances, the reflected light image signal may be orders of magnitude larger than the fluorescence image signal. There are some existing options to compensate for such image signal differences (e.g., by adjusting the fluorophore concentration in the tissue, adjusting the intensity of the excitation light, amplifying the electronic fluorescence image signal when the optical fluorescence image signal is transduced at the image sensor or thereafter, etc.). However, these workarounds may prove insufficient for acquiring an adequate fluorescence image signal. In systems that acquire the reflected light image signal and the fluorescence image signal with the same image sensor, providing sufficient compensation for a relatively weak fluorescence image signal becomes particularly challenging. 
     Furthermore, the reflected light image signal and/or fluorescence image signal may suffer from motion blurring as the result of movement of the imaging system and/or the object being imaged. Such motion blurring, as well as noise in the reflected light and/or fluorescence image signals, may prevent or hamper the visualization of fine details in the fluorescence images. 
     Thus, it is desirable to have medical imaging systems in which low-intensity image signals, such as for example reflected light image signals and/or fluorescence image signals, can be more effectively visualized and presented to a user. 
     SUMMARY OF THE INVENTION 
     According to some embodiments, an adaptive imaging method for generating low light video of an object for medical visualization may include acquiring, with an image acquisition assembly, a sequence of reflected light video frames and/or a sequence of fluorescence video frames depicting the object, assessing relative movement between the image acquisition assembly and the object based on reference video frames that include at least a portion of the acquired sequence of reflected light video frames or a portion of the acquired sequence of fluorescence video frames, adjusting a level of image processing of the reflected light video frames and/or the fluorescence video frames based at least in part on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object, and generating a characteristic low light video output from a quantity of the reflected light video frames and/or a quantity of the fluorescence video frames. In some embodiments, the quantity of the low light video frames for the characteristic low light video output may be based on the adjusted level of image processing of the low light video frames. 
     Such an adaptive imaging method for use in medical imaging may improve visualization of low light image signals from an object while limiting the introduction of image artifacts during relative movement between the image acquisition assembly and the object. In various embodiments, the method may be used when acquiring video of a single low light image signal (e.g. a reflected white light image signal, or a fluorescence image signal) or when acquiring video of a low light image signal while also acquiring video of a relatively higher intensity image signal (e.g. a low light fluorescence image signal and a higher intensity reflected light image signal). In embodiments that include acquiring video of a relatively higher intensity image signal in addition to a low light image signal, the higher intensity image signal may be used as the source of the reference video frames which may provide for improved assessment of relative motion between the image acquisition assembly and the object. In embodiments that include acquiring video of a single low light image signal, that low light image signal may be used as the source of the reference video frames for assessment of relative motion. 
     In various embodiments, relative movement between the image acquisition assembly and the object may be assessed by measuring changes in a plurality of the reference video frames, such as change in pixel intensities. For example, change in pixel intensities may be analyzed by determining a representative pixel intensity for each of a plurality of subregions in the plurality of reference video frames, and characterizing the changes in representative pixel intensity for the subregions in the plurality of the reference video frames. 
     Following the assessment of relative movement between the image acquisition assembly and the object, the level of image processing of the low light video frames may be adjusted by adjusting the quantity of low light video frames from which the characteristic low light video output is generated. In particular, the image processing level may be adjusted based on at least one motion threshold. For instance, adjusting the quantity of low light video frames may include setting the quantity of low light video frames to a first predetermined value if the relative movement (or assessment of relative movement) between the image acquisition assembly and the object is below a first motion threshold, and setting the quantity of low light video frames to a second predetermined value lower than the first predetermined value if the relative movement (or assessment of relative movement) is above the first motion threshold. In some variations, additional motion thresholds may be utilized, such as by setting the quantity of low light video frames to the second predetermined value if the relative movement (or assessment of relative movement) is above the first motion threshold and additionally below a second motion threshold that is higher than the first motion threshold. Additionally, the method may include setting the quantity of low light video frames to a third predetermined value that is lower than the first and second predetermined values if the relative movement (or assessment of relative movement) is above the first and second motion thresholds. In some variations, the quantity of low light video frames may be adjusted or set to a predetermined value by gradually increasing or decreasing the quantity of low light video frames from which the characteristic low light video output is based, over a series of frames of the characteristic fluorescence video output. 
     The characteristic low light video output may be generated using various image processing steps and based on a quantity of low light video frames associated with the adjusted level of image processing. For example, a frame of the characteristic low light video output may be generated by determining a sum of pixel intensities of the quantity of the low light video frames on a region-by-region basis, and optionally additionally dividing the sum of the pixel intensities by the square root of the quantity of the low light video frames combined to generate the frame of the characteristic low light video output. As another example, a frame of the characteristic low light video output may be generated by averaging pixel intensities of the quantity of low light video frames on a region-by-region basis. 
     In some variations, the method may additionally or alternatively include performing other actions based on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object, such as adjusting a low light video frame exposure period and/or controlling a timing scheme of the image acquisition assembly, a visible light source illuminating the object, and/or an excitation light source illuminating the object. 
     The method may further include displaying the characteristic low light video output on a display. Furthermore, the displaying of the low light video output may be generally continuous, as the method in some variations may be performed continuously (e.g., as long as the low light video frames are acquired). 
     Generally, an adaptive imaging system for generating low light video of an object includes an image acquisition assembly configured to acquire a sequence of low light video frames depicting the object, and a processor. The processor may be configured to assess relative movement between the image acquisition assembly and the object based on a portion of reference video frames, which may include at least a portion of the low light video frames and/or a portion of substantially simultaneously acquired higher intensity light video frames, adjust a level of image processing of the low light video frames based at least in part on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object, and generate a characteristic low light video output from a quantity of the low light video frames, wherein the quantity of the low light video frames is based on the adjusted level of image processing of the low light video frames. The system may further include a visible light source that is configured to emit visible light to illuminate the object, and an excitation light source configured to emit excitation light that causes the object to emit fluorescent light. In some variations, the system may further include a controller that controls a timing scheme for the visible light source, the excitation light source, and the image acquisition assembly based at least in part on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object. The system may include a display that is configured to display the characteristic fluorescence video output and/or the reflected light video frames. 
     According to an embodiment, an adaptive imaging system for generating low light fluorescence video of an object includes an image acquisition assembly configured to acquire a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object, and a processor. The processor may be configured to assess relative movement between the image acquisition assembly and the object based on at least a portion of the reflected light video frames, adjust a level of image processing of the fluorescence video frames based at least in part on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object, and generate a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. The system may further include a visible light source that is configured to emit visible light to illuminate the object, and an excitation light source configured to emit excitation light that causes the object to emit fluorescent light. In some variations, the system may further include a controller that controls a timing scheme for the visible light source, the excitation light source, and the image acquisition assembly based at least in part on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object. The system may include a display that is configured to display the characteristic fluorescence video output and/or the reflected light video frames. 
     The processor of the adaptive imaging system may be configured to assess relative movement between the image acquisition assembly and the object by measuring changes in pixel intensities in a plurality of the reference video frames. Based on this relative movement (or assessment of relative movement), the processor may be configured to adjust the level of image processing of the low light video frames, such as by adjusting the quantity of low light video frames from which the characteristic low light video output is generated. More specifically, the processor may set the quantity of low light video frames to a first predetermined value if the relative movement (or assessment of relative movement) between the image acquisition assembly and object is below a first motion threshold, and set the quantity of low light video frames to a second predetermined value if the relative movement (or assessment of relative movement) between the image acquisition assembly and object is above the first motion threshold, wherein the second predetermined value is lower than the first predetermined value. Additionally, the processor may set the quantity of low light video frames to the second predetermined value if the relative movement (or assessment of relative movement) between the image acquisition assembly and the object is above the first motion threshold and below a second motion threshold higher than the first motion threshold and set the quantity of low light video frames to a third predetermined value if the relative movement (or assessment of relative movement) between the image acquisition assembly and the object is above the first and second motion thresholds, the third predetermined value being lower than the first and second predetermined values. In some variations, the processor may adjust or set the quantity of low light video frames to a predetermined value by gradually increasing or decreasing the quantity of low light video frames from which the characteristic low light video output is based, over a series of frames of the characteristic low light video output. 
     The processor may be configured to generate the characteristic low light video output in one or more of various manners, such as by determining a sum of pixel intensities of the quantity of the low light video frames on a region-by-region basis and optionally dividing the sum of pixel intensities by the square root of the quantity of low light video frames, or averaging pixel intensities of the quantity of the low light video frames on a region-by-region basis. 
     Furthermore, in some variations, some or all of the components of the adaptive imaging system may be combined or integrated with other technologies. For example, the adaptive imaging system may further include an image stabilization system that is implemented in hardware, software, or a combination thereof. As another example, the adaptive imaging system described herein may be embodied in an endoscopic imaging system. 
     According to some embodiments, an adaptive imaging method for generating fluorescence video of an object, includes acquiring, with an image acquisition assembly, a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object, assessing relative movement between the image acquisition assembly and the object based on at least a portion of the acquired sequence of reflected light video frames, adjusting a level of image processing of the fluorescence video frames based at least in part on the relative movement between the image acquisition assembly and the object, and generating a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. 
     In any of these embodiments, assessing relative movement between the image acquisition assembly and the object may include measuring changes in pixel intensities in a plurality of the reflected light video frames. In any of these embodiments, assessing relative movement between the image acquisition assembly and the object may include determining a representative pixel intensity for each of a plurality of subregions in the plurality of reflected light video frames, and characterizing the changes in representative pixel intensity for the subregions in the plurality of the reflected light video frames. 
     In any of these embodiments, adjusting the level of image processing of the fluorescence video frames may include adjusting the quantity of fluorescence video frames from which the characteristic fluorescence video output is generated. In any of these embodiments, adjusting the quantity of fluorescence video frames may include setting the quantity of fluorescence video frames to a first predetermined value if the relative movement between the image acquisition assembly and the object is below a first motion threshold. In any of these embodiments, setting the quantity of fluorescence video frames to the first predetermined value may include gradually increasing or decreasing the quantity of fluorescence video frames to the first predetermined value over a series of frames of the characteristic fluorescence video output. 
     In any of these embodiments, adjusting the quantity of fluorescence video frames may include setting the quantity of fluorescence video frames to a second predetermined value if the relative movement between the image acquisition assembly and the object is above the first motion threshold, the second predetermined value being lower than the first predetermined value. In any of these embodiments, setting the quantity of fluorescence video frames to the second predetermined value may include gradually increasing or decreasing the quantity of fluorescence video frames to the second predetermined value over a series of frames of the characteristic fluorescence video output. 
     In any of these embodiments, adjusting the quantity of fluorescence video frames may include setting the quantity of fluorescence video frames to the second predetermined value if the relative movement between the image acquisition assembly and the object is above the first motion threshold and below a second motion threshold higher than the first motion threshold, and setting the quantity of fluorescence video frames to a third predetermined value, if the relative movement between the image acquisition assembly and the object is above the first and second motion thresholds, the third predetermined value being lower than the first and second predetermined values. 
     In any of these embodiments, setting the quantity of fluorescence video frames to the third predetermined value may include gradually increasing or decreasing the quantity of fluorescence video frames to the third predetermined value over a series of frames of the characteristic fluorescence video output. In any of these embodiments, adjusting the quantity of fluorescence video frames may include gradually increasing or decreasing the quantity of fluorescence video frames toward a predetermined value over a series of frames of the characteristic fluorescence video output. In any of these embodiments, generating the characteristic fluorescence video output may include determining a sum of pixel intensities of the quantity of the fluorescence video frames on a region-by-region basis. 
     In any of these embodiments, generating the characteristic fluorescence video output may further include dividing the sum of pixel intensities by the square root of the quantity of the fluorescence video frames. In any of these embodiments, generating the characteristic fluorescence video output from the quantity of the fluorescence video frames may include averaging pixel intensities of the quantity of the fluorescence video frames on a region-by-region basis. In any of these embodiments, the method may further include adjusting a fluorescence video frame exposure period based at least in part on the relative movement between the image acquisition assembly and the object. 
     In any of these embodiments, the method may further include displaying at least one of the characteristic fluorescence video output and the reflected light video frames on a display. In any of these embodiments, the method may further include controlling a timing scheme of a visible light source illuminating the object, an excitation light source illuminating the object, and the image acquisition assembly based at least in part on the relative movement between the image acquisition assembly and the object. In any of these embodiments, the method may be performed continuously. 
     According to some embodiments, an adaptive imaging system for generating fluorescence video of an object, includes an image acquisition assembly configured to acquire a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object, and a processor configured to assess relative movement between the image acquisition assembly and the object based on at least a portion of the reflected light video frames, adjust a level of image processing of the fluorescence video frames based at least in part on the assessed relative movement between the image acquisition assembly and the object, and generate a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. 
     In any of these embodiments, the system may further include a visible light source configured to emit visible light to illuminate the object, and an excitation light source configured to emit excitation light that causes the object to emit fluorescent light. In any of these embodiments, the system may further include a controller that controls a timing scheme for the visible light source, the excitation light source, and the image acquisition assembly based at least in part on the relative movement between the image acquisition assembly and the object. In any of these embodiments, the processor may be configured to assess relative movement by measuring changes in pixel intensities in a plurality of the reflected light video frames. 
     In any of these embodiments, the processor may be configured to adjust the level of image processing of the fluorescence video frames by adjusting the quantity of fluorescence video frames from which the characteristic fluorescence video output is generated. In any of these embodiments, the processor may be configured to adjust the quantity of fluorescence video frames by setting the quantity of fluorescence video frames to a first predetermined value if the relative movement between the image acquisition assembly and object is below a first motion threshold, and setting the quantity of fluorescence video frames to a second predetermined value if the relative movement between the image acquisition assembly and object is above the first motion threshold, the second predetermined value being lower than the first predetermined value. 
     In any of these embodiments, the processor may be configured to adjust the quantity of fluorescence video frames by setting the quantity of fluorescence video frames to the second predetermined value, if the relative movement between the image acquisition assembly and the object is above the first motion threshold and below a second motion threshold higher than the first motion threshold, and setting the quantity of fluorescence video frames to a third predetermined value, if the relative movement between the image acquisition assembly and the object is above the first and second motion thresholds, the third predetermined value being lower than the first and second predetermined values. 
     In any of these embodiments, the processor may be configured to adjust the quantity of fluorescence video frames by gradually increasing or decreasing the quantity of fluorescence video frames toward a predetermined value over a series of frames of the characteristic fluorescence video output. In any of these embodiments, the processor may be configured to generate the characteristic fluorescence video output by performing: (i) determining a sum of pixel intensities of the quantity of the fluorescence video frames on a region-by-region basis, (ii) determining a sum of pixel intensities of the quantity of the fluorescence video frames on a region-by-region basis and dividing the sum of pixel intensities by the square root of the quantity of fluorescence video frames, (iii) averaging pixel intensities of the quantity of the fluorescence video frames on a region-by-region basis, or (iv) a combination thereof. 
     In any of these embodiments, the system may further include a display configured to display at least one of the characteristic fluorescence video output and the reflected light video frames. In any of these embodiments, at least a portion of the imaging system may be embodied in an endoscopic imaging system. In any of these embodiments, the system may further include an image stabilization system implemented in hardware, software, or a combination thereof. 
     According to some embodiments, a computer-implemented, adaptive imaging method for generating fluorescence video of an object, includes receiving a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object, wherein the reflected light video frames and fluorescence video frames are acquired by an image acquisition assembly, assessing relative movement between the image acquisition assembly and the object based on at least a portion of the reflected light video frames, adjusting a level of image processing of the fluorescence video frames based at least in part on the relative movement between the image acquisition assembly and the object, and generating a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. 
     According to some embodiments, an adaptive imaging method for generating fluorescence video of an object, includes acquiring, with an image acquisition assembly, a sequence of reflected light video frames depicting the object, assessing relative movement between the image acquisition assembly and the object based on at least a portion of the acquired sequence of reflected light video frames, adjusting a fluorescence video frame exposure period based at least in part on the relative movement between the image acquisition assembly and the object, and acquiring a sequence of fluorescence video frames using the adjusted fluorescence video frame exposure period. 
     According to some embodiments, an adaptive imaging method for generating enhanced low-intensity light video of an object for medical visualization, includes acquiring, with an image acquisition assembly, a sequence of low light video frames depicting the object, receiving a sequence of reference video frames, assessing relative movement between the image acquisition assembly and the object based on the reference video frames, adjusting a level of image processing of the low light video frames based at least in part on the relative movement between the image acquisition assembly and the object, and generating a characteristic low light video output from a quantity of the low light video frames, wherein the quantity of the low light video frames is based on the adjusted level of image processing of the low light video frames. 
     According to some embodiments, a kit for imaging an object may include a fluorescence imaging agent and the system of any one of the above embodiments. 
     According to some embodiments, a fluorescence imaging agent may include a fluorescence imaging agent for use with the system of any one of the above embodiments, the method of any one of the above embodiments, or the kit of any one of the above embodiments. 
     In any of these embodiments, imaging an object may include imaging an object during blood flow imaging, tissue perfusion imaging, lymphatic imaging, or a combination thereof. 
     In any of these embodiments, blood flow imaging, tissue perfusion imaging, and/or lymphatic imaging may include blood flow imaging, tissue perfusion imaging, and/or lymphatic imaging during an invasive surgical procedure, a minimally invasive surgical procedure, or during a non-invasive surgical procedure. 
     In any of these embodiments, the invasive surgical procedure may include a cardiac-related surgical procedure, or a reconstructive surgical procedure. 
     In any of these embodiments, the cardiac-related surgical procedure may include a cardiac coronary artery bypass graft (CABG) procedure. 
     In any of these embodiments, the CABG procedure may include on pump or off pump. 
     In any of these embodiments, the non-invasive surgical procedure may include a wound care procedure. 
     In any of these embodiments, the lymphatic imaging may include identification of a lymph node, lymph node drainage, lymphatic mapping, or a combination thereof 
     In any of these embodiments, the lymphatic imaging may relate to the female reproductive system. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Features will become apparent to those of ordinary skill in the art by describing in detail exemplary embodiments with reference to the attached drawings in which: 
         FIG. 1A  is an illustrative schematic of an adaptive imaging method for generating low light video of an object according to an embodiment;  FIG. 1B  is an illustrative schematic of an adaptive imaging method for generating fluorescence video of an object according to an embodiment; 
         FIG. 2A  is an illustrative flowchart of one variation of an adaptive imaging method for generating low light video of an object according to an embodiment;  FIG. 2B  is an illustrative flowchart of one variation of an adaptive imaging method for generating fluorescence video of an object according to an embodiment; 
         FIG. 3  is an illustrative schematic of a subsampled image sensor used in one variation of an adaptive imaging method for generating low light video of an object according to an embodiment; 
         FIG. 4  is a table summarizing exemplary characteristics of imaging modes in one variation of an adaptive imaging method for generating low light video of an object; 
         FIG. 5  is an illustrative schematic of variations of generating a characteristic low light video output; 
         FIG. 6A  is an illustrative schematic of one variation of an adaptive imaging system for generating low light video of an object according to an embodiment;  FIG. 6B  is an illustrative schematic of one variation of an adaptive imaging system for generating fluorescence video of an object according to an embodiment; 
         FIG. 7  is an illustrative depiction of one variation of an adaptive imaging system embodied in an endoscopic imaging system according to an embodiment; 
         FIG. 8  is an illustrative schematic of one variation of a light source assembly in an adaptive imaging system according to an embodiment; 
         FIG. 9  is an illustrative schematic of one variation of an image acquisition assembly in an adaptive imaging system according to an embodiment; and 
         FIG. 10  is an illustrative diagram of a range of image quality regimes. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Example embodiments will now be described more fully hereinafter with reference to the accompanying drawings; however, they may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey exemplary implementations to those skilled in the art. Various devices, systems, methods, processors, kits and imaging agents are described herein. Although at least two variations of the devices, systems, methods, processors, kits and imaging agents are described, other variations may include aspects of the devices, systems, methods, processors, kits and imaging agents described herein combined in any suitable manner having combinations of all or some of the aspects described. 
     Generally, corresponding or similar reference numbers will be used, when possible, throughout the drawings to refer to the same or corresponding parts. 
     Spatially relative terms, such as “beneath”, “below”, “lower”, “above”, “upper”, and the like, may be used herein for ease of description to describe one element or feature&#39;s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the exemplary term “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. 
     In various embodiments, with reference to a particular use environment (e.g., imaging modality, clinical application, or a combination thereof), “low light video” comprises video wherein the signal to noise ratio (SNR) of the imaged light is relatively low enough such that it may cause image noise to interfere with the ability to clearly visualize a target feature in the image. In various embodiments, the minimum size of a clinically important target feature may vary according to the use environment, with a lower minimum target feature size generally requiring a higher SNR to yield a given level of feature visibility than a higher minimum target feature size. As shown in  FIG. 10 , varying video image quality regimes with very good visibility of a target feature, moderate visibility, and poor visibility may generally be expected to depend on the SNR, the minimum target feature size for a given use environment or a combination thereof. For example, the methods and systems for adaptive imaging for low light signal enhancement, as described herein in accordance with the various embodiments, may facilitate imaging low light video within the moderate visibility or poor visibility image quality regimes. According to some embodiments, the methods and systems may allow for user input to determine whether to apply the adaptive imaging for a given use environment and an anticipated associated image quality regime. In some variations, the adaptive imaging may be activated or deactivated automatically depending on the user-indicated use environment. 
     Generally, the methods and systems described herein may be used to generate real-time enhanced low light videos (including, for example, reflected light videos and/or fluorescence videos), such as for use in applications including imaging of tissue (e.g., during endoscopic examinations, surgical procedures (e.g., minimally invasive), open field imaging, and/or other imaging performed with medical imaging systems, including handheld imaging systems). In some embodiments, the low light video may comprise reflected light video that may be based on visible spectrum light that illuminates and subsequently is reflected from tissue to be visualized. In some embodiments, the low light video may comprise fluorescence light video that may be based on fluorescent light that is emitted by fluorophores located in the tissue to be visualized, after the fluorophores are excited by excitation spectrum light. In some embodiments, a higher intensity light video may be acquired in addition to the low light video. In particular, the adaptive imaging methods and systems described herein may be configured to enhance a low light image signal such as by compensating for signal noise and/or for motion blur. In some embodiments, the adaptive imaging methods and systems described herein may be configured to compensate for a low light image signal that is relatively low-intensity compared to a high-intensity reference image signal, and/or compensate for motion blur and/or signal noise. 
     Adaptive Imaging Method 
       FIG. 1  illustrates a schematic of an adaptive imaging method for low light signal enhancement in medical imaging according to an embodiment. As shown in  FIG. 1A , an example of an adaptive imaging method  100  for generating fluorescence video of an object may include: acquiring, with an image acquisition assembly, a sequence of low light video depicting the object  110 ; assessing relative movement between the image acquisition assembly and the object  120  based on reference video frames comprising at least a portion of the acquired sequence of low light video frames and/or a portion of a substantially simultaneously acquired sequence of higher intensity light video frames; adjusting a level of image processing of the low light video frames  140  based at least in part on the relative movement between the image acquisition assembly and the object; and generating a characteristic low light video output from a quantity of the low light video frames  160 , wherein the quantity of the low light video frames is based on the adjusted level of image processing of the low light video frames. 
     In another variation, an adaptive imaging method for generating fluorescence video of an object may include: acquiring, with an image acquisition assembly, a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object; assessing relative movement between the image acquisition assembly and the object based on at least a portion of the acquired sequence of reflected light video frames; adjusting a level of image processing of the fluorescence video frames based at least in part on the relative movement between the image acquisition assembly and the object; and generating a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. 
     In some embodiments, the method may include controlling a timing scheme of one or more light sources and the image acquisition assembly, based at least in part on the relative movement between the image acquisition assembly and the object. The method may further include displaying at least one of the characteristic low light video output (e.g., fluorescence video output) and the reference video frames  170  (e.g., reflected light video frames) on a display. In some variations, the method may be performed continuously throughout acquisition of the low light video frames (e.g., reflected light video frames, or fluorescence video frames). 
     In an embodiment, as shown in  FIG. 1B , an example of an adaptive imaging method  1100  for generating fluorescence video of an object may include: acquiring, with an image acquisition assembly, a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object  1110 ; assessing relative movement between the image acquisition assembly and the object  1120  based on at least a portion of the acquired sequence of reflected light video frames; adjusting a level of image processing of the fluorescence video frames  1140  based at least in part on the relative movement between the image acquisition assembly and the object; and generating a characteristic fluorescence video output from a quantity of the fluorescence video frames  1160 , wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. The method may include controlling a timing scheme of one or more light sources and the image acquisition assembly, based at least in part on the relative movement between the image acquisition assembly and the object. The method may further include displaying at least one of the characteristic fluorescence video output and the reflected light video frames  1170  on a display. In some variations, the method may be performed continuously throughout acquisition of the reflected light video frames and fluorescence video frames. 
     Acquiring Image Sequences 
     According to an embodiment, the method may include acquiring a sequence of low light video frames depicting the object to be visualized (e.g., tissue), with the use of an image acquisition assembly including at least one image sensor. Such acquisition may include illuminating the object with illumination light (e.g., light in the visible light spectrum) and/or excitation light. The low light video frames may comprise reflected light video frames that may be obtained with the image acquisition assembly receiving illumination light that is reflected from the tissue. The reflected light video frames may include color images and/or grayscale images, depending on the kind of image sensors in the image acquisition assembly, as further described below. Additionally, or alternatively, the low light video frames may comprise fluorescence video frames that may be obtained with the image acquisition assembly receiving fluorescence light that is emitted from intrinsic and/or extrinsic fluorophores (e.g., a fluorescence imaging agent introduced into the object) that are present in the tissue and excited by the excitation light. In addition to the sequence of low light video frames, the method may include acquiring a sequence of reference video frames comprising higher light intensity video frames. 
     According to an embodiment, the method may include acquiring sequences of reflected light video frames and fluorescence video frames depicting the object to be visualized (e.g., tissue), with the use of an image acquisition assembly including at least one image sensor. Such acquisition may include illuminating the object with illumination light (e.g., light in the visible light spectrum) and excitation light. Reflected light video frames that may be obtained with the image acquisition assembly receiving illumination light that is reflected from the tissue. The reflected light video frames may include color images and/or grayscale images, depending on the kind of image sensors in the image acquisition assembly, as further described below. Fluorescence video frames that may be obtained with the image acquisition assembly receiving fluorescence light that is emitted from intrinsic and/or extrinsic fluorophores (e.g., a fluorescence imaging agent introduced into the object) that are present in the tissue and excited by the excitation light. 
     In some variations, the low light video frames may be fluorescence video frames and the reference video frames may be higher light intensity reflected light video frames. The reflected light video frames and the fluorescence video frames may be acquired substantially in parallel or simultaneously, and in real-time. For example, the reflected light image signal and the fluorescence image signal may be acquired with respective image sensors. As another example, in variations in which the same single image sensor is used to acquire both reflected light video frames and fluorescence video frames, the acquisition of video frames may be performed according to a timing scheme. This timing scheme may enable separation of the image signal associated with the reflected light and the image signal associated with the fluorescence emission light. In particular, the timing scheme may involve illuminating the object with illumination light and excitation light according to a pulsing scheme, and processing the reflected light image signal and fluorescence image signal with a processing scheme, wherein the processing scheme is synchronized and matched to the pulsing scheme (e.g., via a controller) to enable separation of the two image signals in a multiplexed manner. Examples of such pulsing and image processing schemes have been described in U.S. Pat. No. 9,173,554, filed on Mar. 18, 2009 and titled “IMAGING SYSTEM FOR COMBINED FULL-COLOR REFLECTANCE AND NEAR-INFRARED IMAGING,” the contents of which are incorporated in their entirety by this reference. However, other suitable pulsing and image processing schemes may be used to acquire reflected light video frames and fluorescence video frames simultaneously. 
     In an embodiment, as the low light video frames and/or the reference video frames are acquired, at least a portion of them may be stored (e.g., in a memory unit) for record-keeping purposes and/or retrieval for analysis during other aspects of the method, as described below. 
     In an embodiment, as the reflected light video frames and the fluorescence video frames are acquired, at least a portion of them may be stored (e.g., in a memory unit) for record-keeping purposes and/or retrieval for analysis during other aspects of the method, as described below. 
     Assessing Relative Movement 
     According to some embodiments, the method may include assessing the relative movement between the image acquisition assembly and the object based on the reference video frames  120 . The assessment of such relative movement may, for example, provide a parameter, where the parameter can be used to determine the manner in which the low light video frames should be processed and/or acquired in order to appropriately increase sensitivity or reduce noise in the low light imaging. The relative movement may result, for example, from unsteady handling of the image acquisition assembly (e.g., when the image acquisition assembly is located in a handheld laparoscope or other handheld imaging system), or movement of a patient being imaged. The relative movement may be better represented in the reference video frames, since the reference video frames may preferably be acquired in real-time with relatively higher light intensity and relatively low latency. In some variations, however, a higher light intensity video signal may not be available and the reference video frames may instead comprise frames of the acquired low light video frames. 
     According to some embodiments, the method may include assessing the relative movement between the image acquisition assembly and the object based on the reflected light video frames. The assessment of such relative movement may, for example, provide a parameter, where the parameter can be used to determine the manner in which the fluorescence video frames should be processed and/or acquired in order to appropriately increase sensitivity or reduce noise in the fluorescence imaging. The relative movement may be better represented in the reflected light video frames, since the reflected light video frames may be acquired in real-time with relatively higher light intensity and relatively low latency. 
     In some variations, assessing relative movement between the image acquisition assembly and the object  120  may include measuring changes in pixel intensities in a plurality of the reference video frames. The pixel intensities in the reference video frames may be measured, for example, from luminance grayscale images based on the reference video frames. In variations in which the reference video frames are color images or white light images (e.g., acquired with one or more image sensors with a color filter array such as a Bayer pattern filter), the method may include generating luminance grayscale images from the luminance (brightness) components of the reference video frames. In variations in which the reference video frames are acquired with a non-color image sensor (e.g., an image sensor without a color filter array), the reference video frames may need not to be converted into a separate luminance image. 
     In some variations, assessing relative movement between the image acquisition assembly and the object may include measuring changes in pixel intensities in a plurality of the reflected light video frames. The pixel intensities in the reflected light video frames may be measured, for example, from luminance grayscale images based on the reflected light video frames. In variations in which the reflected light video frames are color images or white light images (e.g., acquired with one or more image sensors with a color filter array such as a Bayer pattern filter), the method may include generating luminance grayscale images from the luminance (brightness) components of the reflected light video frames. In variations in which the reflected light video frames are acquired with a non-color image sensor (e.g., an image sensor without a color filter array), the reflected light video frames may need not to be converted into a separate luminance image. 
     In an exemplary embodiment shown in  FIG. 2A , after acquiring low light video frames ( 210 ), assessing the relative movement may include determining a representative pixel intensity for one or more subregions in the reference video frames ( 222 ), wherein the reference video frames may comprise at least a portion of the low light video frames and/or a portion of substantially simultaneously acquired higher intensity light video frames. In particular, each reference video frame may include a plurality of subregions, where each subregion includes a group of pixels. For example, as shown in  FIG. 3 , a video frame  340  may include subregions  342   a  and  342   b,  where each subregion  342   a  or  342   b  includes a group of pixels  344  arranged in a cluster (e.g., 1024 pixels arranged in a 32×32 grid, or 100 pixels arranged in a 10×10 grid as depicted in  FIG. 3 , etc.). The representative pixel intensity for each of one or more subregions may be calculated as the average (e.g., mean) intensity of the group of pixels in the subregion, median intensity of the group of pixels in the subregion, or other manner that is characteristic of the overall pixel intensity of the subregion. Utilizing a representative pixel intensity for multi-pixel subregions may reduce computational complexity of the motion assessment and/or reduce the sensitivity to the resolution of the motion assessment. Alternatively, one or more of the subregions may include a single individual pixel, such as in instances in which computational complexity is less of a concern or more detailed/higher resolution intensity information is desired for assessing relative movement. 
     In an embodiment, after acquiring reflected light and fluorescence video frames, assessing the relative movement may include determining a representative pixel intensity for one or more subregions in the reflected light video frames. In particular, each reflected light video frame may include a plurality of subregions, where each subregion includes a group of pixels as described herein. 
     In an exemplary embodiment shown in  FIG. 2B , after acquiring reference reflected light video frames and low light fluorescence video frames ( 1210 ), assessing the relative movement may include determining a representative pixel intensity for one or more subregions in the reflected light video frames ( 1222 ). In particular, each reflected light video frame may include a plurality of subregions, where each subregion includes a group of pixels. For example, as shown in  FIG. 3 , a video frame  340  may include subregions  342   a  and  342   b,  where each subregion  342   a  or  342   b  includes a group of pixels  344  arranged in a cluster (e.g., 1024 pixels arranged in a 32×32 grid, or 100 pixels arranged in a 10×10 grid as depicted in  FIG. 3 , etc.). The representative pixel intensity for each of one or more subregions may be calculated as the average (e.g., mean) intensity of the group of pixels in the subregion, median intensity of the group of pixels in the subregion, or other manner that is characteristic of the overall pixel intensity of the subregion. Utilizing a representative pixel intensity for multi-pixel subregions may reduce computational complexity of the motion assessment and/or reduce the sensitivity to the resolution of the motion assessment. Alternatively, one or more of the subregions may include a single individual pixel, such as in instances in which computational complexity is less of a concern or more detailed/higher resolution intensity information is desired for assessing relative movement. Although  FIG. 3  depicts rectangular or grid-like subregions, the subregions may have any suitable shape. The subregions may be substantially identical in size and shape, though in some variations, some subregions may be different in size or shape. For example, if some areas of the video frames are identified as more important (e.g., depict an object of interest instead of background) and it is desirable to assess relative motion based on more detailed information for those particularly important areas, subregions in the particularly important areas may be smaller than less-important regions. All of the subregions may be considered in the assessment of relative motion or, alternatively, only a subset of one or more of the subregions may be considered, for example to reduce computational complexity and/or to focus assessment of relative motion on a particular region of interest. For example, in some variations, one of the subsets of alternating subregions  342   a  and  342   b,  may be omitted from consideration in the assessment of relative motion. 
     In an embodiment, assessing the relative movement may further include characterizing the change in the representative pixel intensity for the subregions in the reference video frames. In an embodiment, assessing the relative movement may further include characterizing the change in the representative pixel intensity for the subregions in the reflected light video frames. The characterization may be quantitative (e.g., a numerical value describing the magnitude of relative movement between the image acquisition assembly and the object). At least two sequential video frames may be analyzed (e.g., to characterize how the representative pixel intensities for the subregions have changed between an immediately prior video frame to a current frame, or between non-adjacent frames representing endpoints of a multi-frame time period of interest). For instance, in the exemplary embodiment shown in  FIG. 2A , assessing the relative movement may further include determining the change in representative pixel intensity for the subregions  224  and determining a statistical measure of the change in representative pixel intensity for the subregions  226 . The statistical measure is representative of the assessed relative motion between the image acquisition assembly and the object. For example, to characterize the relative movement of the image acquisition assembly and object over the course of two adjacent reference video frames (or two adjacent reflected light video frames), step  224  may include determining the difference between the representative pixel intensity for a subregion in a current video frame and the representative pixel intensity for the same subregion in a previous video frame, and repeating this determination for all considered subregions. Subsequently, determining a statistical measure  226  may include, for example, calculating the standard deviation of the differences determined in block  224 . As another example, determining a statistical measure  226  may include calculating the average (mean, etc.) of the differences determined in block  224 . Other statistical measures of the change in representative pixel intensity for the subregions may additionally or alternatively be used to characterize the change in representative pixel intensity and arrive at a measure of the assessed relative motion between the image acquisition assembly and the object. 
     In some variations, assessing relative movement between the image acquisition assembly and the object being imaged may include subsampling the pixel intensities of the reflected light video frames. In particular, the relative movement may be assessed based on pixel intensities of a selected portion of the subregions (and accordingly, a selected number of the pixels). Such subsampling may reduce the overall computational complexity of the motion assessment. For example, as shown in  FIG. 3 , the relative movement may be based on a subsampling of subregions  342   a  that are distributed in an alternating checkerboard pattern. In this example, information from subregions  342   a  (shaded in  FIG. 3 ) may be utilized in assessing relative motion, while information from subregions  342   b  (unshaded in  FIG. 3 ) may be ignored. However, the motion assessment may incorporate any suitable sub sampling scheme (e.g., sampling different portions of the video frames, such as one-third, two-third, or another fraction of the subregions). 
     The method may additionally or alternatively include any other suitable motion-estimation algorithms using the pixel intensities and/or other characteristics of the reference video frames (e.g., based on a color component such as red, green, or blue of reference reflected light video frames, based on chroma values of reference reflected light video frames, etc.). In some variations, the method may additionally or alternatively include motion-estimation algorithms using the pixel intensities and/or other characteristics of the low light video frames (though, for example, using low light fluorescence video frames instead of reference reflected light video frames for motion-estimation may be less reliable in some circumstances). Additionally, in some variations in which the video frames include voxels, the method may include implementing the above-described motion-estimation algorithms with respect to characteristics of voxels (e.g., voxel intensity) instead of or in addition to characteristics of pixels (e.g., pixel intensity). Furthermore, in some variations the method may additionally or alternatively include receiving information from a gyroscope or other hardware configured to detect motion of the image acquisition assembly, and utilizing this information to assess movement of the image acquisition assembly. 
     The method may additionally or alternatively include any other suitable motion-estimation algorithms using the pixel intensities and/or other characteristics of the reflected light video frames (e.g., based on a color component such as red, green, or blue of reflected light video frames, based on chroma values of the reflected light video frames, etc.). In some variations, the method may additionally or alternatively include motion-estimation algorithms using the pixel intensities and/or other characteristics of the fluorescence frames (though, for example, using fluorescence video frames instead of the reflected light video frames for motion-estimation may be less reliable in some circumstances). 
     Between iterations, one or more of the values calculated during motion assessment may be stored in memory for future use. For example, the representative pixel intensities for the subregions in a current video frame may be stored for use in a future iteration of the calculations, to be used as the representative pixel intensities for the subregions in a previous video frame. 
     Adjusting the Level of Image Processing 
     Generally, the method may include selecting an imaging mode suitable for different amounts of relative movement between the image acquisition assembly and the object being imaged. In some variations, upon selection of an imaging mode, the method includes adjusting a level of image processing of the low light video frames  140  (e.g., fluorescence video frames) based on the relative movement (or assessment of relative movement) between the image acquisition assembly and the object being imaged. The level of image processing may be adjusted in order to increase sensitivity and/or reduce noise in the characteristic fluorescence video output. 
     For instance, upon selection of an imaging mode, adjusting the level of image processing of the low light video frames may include adjusting the quantity of low light video frames from which the characteristic low light video output is generated. At least some of the imaging modes may correspond to a respective, predetermined plural quantity of low light video frames that may be combined (as described in further detail below) to generate a characteristic low light video output with amplified or enhanced low light data from the combined low light video frames. However, more motion artifacts (e.g., motion blur) may appear in the characteristic low light video output if there is a significant amount of relative movement between the image acquisition assembly and the object throughout the combined low light video frames. Thus, in order to reduce motion artifacts in the characteristic low light video output, the quantity of low light video frames that is used to generate each frame of the characteristic low light video output may generally be inversely related to the degree of relative movement between the image acquisition assembly and the object being imaged. 
     For example, as shown in  FIG. 4 , a “low-motion mode” may correlate to a relatively high quantity of low light video frames (e.g., fluorescence video frames) that are combined to generate a frame of the low light video output during the low-motion mode. This low-motion mode may be suitable, for instance, when there are no or negligible changes in relative positions of the image acquisition assembly and the object being imaged. A “moderate-motion mode” may correlate to a moderate quantity of low light video frames that are combined to generate a frame of the low light video output during the moderate-motion mode. This moderate-motion mode may be suitable, for instance, when there are small or slight changes in relative positions of the image acquisition assembly and the object being imaged. A “high-motion mode” may correlate to a relatively low quantity (e.g., one) of low light video frames that are combined to generate a frame of the low light video output during the high-motion mode. This high-motion mode may be suitable, for instance, when there are major changes in relative positions of the image acquisition assembly and the object being imaged, and when it is desirable to avoid smearing or motion blur of the fluorescence image features and/or desirable to reduce the time lag in the displayed fluorescence video output. 
     Although  FIG. 4  depicts an exemplary embodiment of the method with three imaging modes, other embodiments of the method may have fewer (e.g., 2) or more (e.g., 4, 5, or 6, etc.) imaging modes following a similar trend with respect to quantity of low light video frames (e.g., fluorescence video frames) that are combined to generate the characteristic low light video output (e.g., fluorescence video output). 
     Referring to  FIG. 2A , after a measure representative of the assessed relative movement between the image acquisition assembly and the object is determined ( 226 ), the measure may be compared to one or more motion thresholds. Based on these comparisons, the particular imaging mode may be set to low-motion mode ( 252 ), moderate-motion mode ( 254 ), or high-motion mode ( 256 ). Subsequently, adjusting the level of image processing of the low light video frames (e.g., fluorescence video frames) may include adjusting the quantity of low light video frames from which the characteristic low light video output is generated, according to the set imaging mode. 
     More specifically, if the measure representative of the assessed relative movement is below a low-motion threshold ( 242 ), then the method may set the imaging system to a low-motion mode ( 252 ) such that adjusting the quantity of low light video frames involves setting the quantity of low light video frames (from which the characteristic low light video output is generated) to a predetermined high value. Additionally, if the measure is above the low-motion threshold ( 242 ), then the measure may be compared to a high-motion threshold ( 244 ). If the measure is above the low-motion threshold but below the high-motion threshold ( 244 ), then the method may set the imaging system to a moderate-motion mode ( 254 ) such that adjusting the quantity of low light video frames involves setting the quantity of low light video frames to a predetermined moderate value that is lower than the predetermined high value. If the measure is above the high-motion threshold ( 244 ), then the method may set the imaging system to a high-motion mode ( 256 ) such that adjusting the quantity of low light video frames involves setting the quantity of low light video frames (from which the characteristic low light video output is generated) to a predetermined low value that is lower than the predetermined moderate and high values. 
     In some variations, when the imaging system is set to a new imaging mode, the method may include gradually increasing or decreasing the quantity of low light video frames (from which the characteristic low light video output is generated) to the predetermined value associated with the new imaging mode. For instance, the gradual increasing or decreasing may occur over a series of frames of the characteristic low light video output, as defined by a predetermined number of frames or a predetermined period of time. This gradual change may help provide a smooth visual transition between previously-selected and currently-selected imaging modes. For example, when transitioning to the low-motion mode, setting the quantity of low light video frames to the predetermined high value may include gradually increasing the quantity of low light video frames. Similarly, when transitioning from the low-motion or the high-motion imaging mode to the moderate-motion mode, setting the quantity of low light video frames to the predetermined mid value may include gradually decreasing or increasing, respectively, the quantity of low light video frames. When transitioning to the high-motion mode, setting the quantity of low light video frames to the predetermined low value may include gradually decreasing the quantity of low light video frames in some variations. However, in some variations, it may be particularly desirable to immediately set the quantity to the predetermined low value when transitioning to the high-motion mode, to reduce motion artifacts in the low light video output when they are most likely to occur. 
     Generating a Characteristic Image 
     The method may include generating a characteristic low light video output  160  (e.g., a fluorescence video output) from the quantity of low light video frames (e.g., fluorescence video frames) based on the adjusted level of image processing. Generally, the quantity of low light video frames may be combined into a frame of the characteristic low light video output such that the low light video output visualizes amplified and/or de-noised low light image data from the combined low light video frames. 
     As shown in  FIG. 5 , given a quantity N of low light video frames  550  (e.g., fluorescence video frames), the N frames may be combined in any one or more of several manners to generate a particular frame of the characteristic low light video output. In one variation, generating the characteristic low light video output  560  may include determining a sum of pixel intensities of the N low light video frames  550  on a region-by-region basis  562 . Summing the pixel intensities may increase the low light signal intensity in the characteristic low light video output. 
     In another variation, generating the characteristic low light video output  560  (e.g., fluorescence video output) may include determining a sum of pixel intensities of the N low light video frames  550  on a region-by-region basis  564  (similar to  562 ) and further dividing the sum of the pixel intensities by a scaling factor  566 , such as the square root of the number of video frames N. Other suitable scaling factors besides solely the square root of N may additionally or alternatively be used to scale the sum of the pixel intensities. This variation may partially increase the low light signal intensity in the characteristic low light video output, while limiting or eliminating any corresponding increase in the intensity of the image noise. 
     In yet another variation, generating the characteristic low light video output  560  may include averaging the pixel intensities of the quantity N of low light video frames  568 . This may preserve the low light signal intensity in the characteristic low light video output, but may additionally reduce the intensity of image noise (that is, increase the signal-to-noise ratio). 
     In other variations, the characteristic low light video output may be generated from a selected quantity of low light video frames  550  in other suitable manners. 
     Other Low Light Imaging Adjustments 
     In another variation, at least some of the imaging modes may additionally or alternatively correspond to a respective low light video frame (e.g., a respective fluorescence video frame) exposure period. Longer exposure periods allow for an amplified low light image signal, but are also associated with a higher risk of motion artifacts resulting from relative movement between the image acquisition assembly and the object throughout the exposure period. Thus, in order to reduce motion artifacts in the characteristic low light video output, the duration of the exposure period may generally be inversely related to the degree of relative movement between the image acquisition assembly and the object being imaged. For example, as shown in  FIG. 4 , a low-motion mode imaging may be associated with a relatively high exposure (i.e., relatively longer period of time), a moderate-motion imaging mode may be associated with a moderate exposure, and a high-motion imaging mode may be associated with a relatively low exposure (i.e., relatively shorter period of time). Although  FIG. 4  depicts an exemplary embodiment of the method with three imaging modes, other embodiments of the method may have fewer (e.g., 2) or more (e.g., 4, 5, or 6, etc.) imaging modes following a similar trend with respect to the low light video frame exposure period. 
     The adjustment of low light video frame exposure period may be supplemental to the multi-frame low light image processing described above for the different imaging modes. Alternatively, the adjustment of low light video frame exposure period may be performed without the multi-frame low light image processing, such that the method includes adjusting a low light video frame exposure period based at least in part on the relative movement between the image acquisition assembly and the object being imaged, and acquiring a sequence of low light video frames using the adjusted low light video frame exposure period. 
     Additionally, similar to the above-described gradual transition in the quantity of low light video frames being used for generating the characteristic low light video output, the transition between the imaging modes may involve gradually lengthening or shortening the low light video frame exposure period over a series of frames of the characteristic low light video output. 
     In other variations, such as for variations in which reference light video frames (e.g., reflected light video frames) and low light video frames (e.g., fluorescence video frames) are acquired with a single image sensor, the method may further include controlling a timing scheme of a visible light source that is illuminating the object, an excitation light source that is illuminating the object, and the image acquisition assembly. The timing scheme may be controlled based at least in part on the relative movement between the image acquisition assembly and the object. For instance, in one example, the timing scheme in a moderate-motion imaging mode may involve a repeated pattern of illumination and image processing for two successive reflective light video frames followed by one fluorescence light video frame. In a low-motion mode, the timing scheme may involve a repeated pattern of illumination and image processing for one reflective light video frame followed by two fluorescence light video frames which facilitates fluorescence image signal amplification and/or noise reduction in the characteristic fluorescence video output. Similar adjustments to the number of frames for fluorescence image acquisition may be performed for other variations of timing sequences. 
     Another example may be implemented in instances where two color component signals (e.g., green and blue) are continuously read on two of three channels on an image sensor while a third color component signal (e.g., red) and a fluorescence signal are alternatively read on the third channel of the image sensor. In this example, the timing scheme in a moderate-motion imaging mode may involve a repeated pattern of the illumination and image processing for two successive video frames with the third color component and one video frame with the fluorescence (e.g., GB+R, GB+R, GB +FL). In a low-motion imaging mode, the timing scheme may involve a repeated pattern of illumination and image processing for one video frame with the third color component and two successive video frames with the fluorescence (e.g., GB+R, GB+FL, GB+FL) which facilitates fluorescence image signal amplification and/or noise reduction in the characteristic fluorescence video output. Similar adjustments to the number of frames for fluorescence image acquisition may be performed for other variations of timing sequences. 
     In yet another variation, the method may include applying image stabilization technology to compensate for at least some ranges of motion or relative movement between the image acquisition assembly and the object (e.g., due to unsteady handling of the image acquisition assembly). The image stabilization technology may, for example, be implemented in hardware and/or image processing software, either as part of the imaging system or as a separate plug-in electronic image stabilization module. For instance, the level of image processing of the low light video frames (e.g., fluorescence video frames) may be adjusted based on an assessment of the residual evidence of relative movement that remains in at least a portion of the acquired sequence of reference video frames (e.g., reflected light video frames) after compensation by the image stabilization technology (e.g., instead of based on the actual, greater amount of relative movement determinable from video frames without compensation by the image stabilization technology). 
     Displaying 
     As shown in  FIG. 1A , the method may include displaying at least one of the characteristic low light video output (e.g., fluorescence video output) and the reference video frames  170  (e.g., reflected light video frames) on a display (e.g., monitor or screen). In some instances (e.g., based on operator-selected settings), the method may include displaying both the characteristic low light video output and the sequence of reference video frames, either side-by-side or overlaid or otherwise merged. If the characteristic low light video output and the sequence of reference video frames are merged, then the low light video output may be displayed in high contrast to the reference video frames such as in a display color that is not commonly present in the body (e.g., bright green, purple). Similarly, the method may include displaying low light video frames acquired using an adjusted low light video frame exposure period, and/or the reference video frames either side-by-side or merged as described above. 
     The method may include storing or printing at least some frames of the characteristic low light video output, acquired low light video frames, and/or acquired reference video frames. For instance, desired video frames may be selected by the user via a user interface for storing in a memory unit, display, printing, etc. 
     Adaptive Imaging System 
     Generally, as shown in  FIG. 6A , an example of an adaptive imaging system  600  for generating low light video of an object  602  (e.g., a tissue region of interest) may include: an image acquisition assembly  620  with at least one image sensor  622  configured to acquire a sequence of low light video frames depicting the object; and a processor  630 . The processor  630  is configured to assess relative movement between the image acquisition assembly  620  based on reference video frames comprising at least a portion of the low light video frames and/or a portion of a substantially simultaneously acquired sequence of higher intensity video frames, adjust a level of processing of the low light video frames based at least in part on the relative movement between the image acquisition assembly  620  and the object  602 , and generate a characteristic low light video output from a quantity of the low light video frames, wherein the quantity of the low light video frames is based on the adjusted level of image processing of the low light video frames. In other variations, the processor  630  may be configured to perform aspects of the method  100  described above. 
     In accordance with some embodiments, as shown in  FIG. 6B , an example of an adaptive imaging system  1600  for generating fluorescence video of an object  1602  (e.g., a tissue region of interest) may include: an image acquisition assembly  1620  with at least one image sensor  1622  configured to acquire a sequence of reflected light video frames and a sequence of fluorescence video frames depicting the object; and a processor  1630 . The processor  1630  is configured to assess relative movement between the image acquisition assembly  1620  based on at least a portion of the reflected light video frames, adjust a level of processing of the fluorescence video frames based at least in part on the relative movement between the image acquisition assembly  1620  and the object  1602 , and generate a characteristic fluorescence video output from a quantity of the fluorescence video frames, wherein the quantity of the fluorescence video frames is based on the adjusted level of image processing of the fluorescence video frames. In other variations, the processor  1630  may be configured to perform aspects of the method  1100  described above. 
     In some variations, at least part of the adaptive imaging system may be embodied in an endoscopic imaging system, such as for minimally-invasive procedures. For example, as shown in  FIG. 7 , an endoscopic imaging system  700  may include an illuminator  702  with a light source assembly configured to provide visible light and/or fluorescence excitation light to a surgical laparoscope  704  via a light guide  706  that is connected to the illuminator  702  via a light guide port  708 . A processor  710  and/or controller  720  may, in some variations, be within the same housing as the illuminator  702 , as shown in  FIG. 7 , and may be configured to perform at least some of the aspects of the method  100  described above. An image acquisition assembly  712  may receive signals via connection to the laparoscope  704 , and may pass acquired images to the processor  710  via connection to the processor  710  such as through port  714 . Certain aspects of the light source assembly, image acquisition assembly, processor, and/or controller may be similar to those described in more detail below. 
     Light Source Assembly 
     As shown in the schematic of  FIG. 6A , the imaging system  600  may include a light source assembly  610  including a visible light source  612  that emits visible light (e.g., full spectrum visible light, narrow band visible light, or other portions of the visible light spectrum) and/or an excitation light source  614  that emits excitation light for exciting fluorophores in the object  602  and causing fluorescence emission. 
     The visible light source  612  is configured to emit visible light for illumination of the object to be imaged. In some variations, the visible light source may include one or more solid state emitters, such as LEDs and/or laser diodes. For example, the visible light source may include blue, green, and red (or other color components) LEDs or laser diodes that in combination generate white light illumination. These color component light sources may be centered around the same wavelengths around which the image acquisition assembly (described further below) is centered. For example, in variations in which the image acquisition assembly includes a single chip, single color image sensor having an RGB color filter array deposited on its pixels, the red, green, and blue light sources may be centered around the same wavelengths around which the RGB color filter array is centered. As another example, in variations in which the image acquisition assembly includes a three-chip, three-sensor (RGB) color camera system, the red, green, and blue light sources may be centered around the same wavelengths around which the red, green, and blue image sensors are centered. 
     The excitation light source  614  is configured to emit excitation light suitable for exciting intrinsic fluorophores and/or extrinsic fluorophores (e.g., a fluorescence imaging agent introduced into the object) located in the object being imaged. The excitation light source  614  may include, for example, one or more LEDs, laser diodes, arc lamps, and/or illuminating technologies of sufficient intensity and appropriate wavelength to excite the fluorophores located in the object being imaged. For example, the excitation light source may be configured to emit light in the near-infrared (NIR) waveband (such as, for example, approximately 805 nm light), though other excitation light wavelengths may be appropriate depending on the application. 
     The light source assembly  610  may further include one or more optical elements that shape and/or guide the light output from the visible light source  612  and/or excitation light source  614 . The optical components may include one or more lenses, mirrors (e.g., dichroic mirrors), light guides and/or diffractive elements, e.g., so as to help ensure a flat field over substantially the entire field of view of the image acquisition assembly  620 . For example, as shown in the schematic of  FIG. 8 , the output  824  from a laser diode  822  (providing visible light or excitation light) may be passed through one or more focusing lenses  826 , and then through a light guide  828 . The light may be further passed through an optical diffractive element  832  (e.g., one or more optical diffusers). Power to the laser diode  822  may be provided by, for example, a high-current laser driver and may optionally be operated in a pulsed mode during the image acquisition process according to a timing scheme. An optical sensor such as a solid state photodiode  830  may be incorporated into the light source assembly and may sample the illumination intensity produced by one or more of the light sources, via scattered or diffuse reflections from the various optical elements. 
     Image Acquisition Assembly 
     The image acquisition assembly  620  may acquire reflected light video frames based on visible light that has reflected from the object, and/or fluorescence video frames based on fluorescence emitted by fluorophores in the object that are excited by the fluorescence excitation light. As shown in  FIG. 9 , the image acquisition assembly  620  may acquire images using a system of optics (e.g., one or more lenses  946   a,  one or more filters  948 , one or more mirrors  950 , beam splitters, etc.) to collect and focus reflected light and/or fluorescent light  942  onto an image sensor assembly  944 . The image sensor assembly  944  may include at least one solid state image sensor. The one or more image sensors may include, for example, a charge coupled device (CCD), a CMOS sensor, a CID, or other suitable sensor technology. In one variation, the image sensor assembly  944  may include a single chip, single image sensor (e.g., a grayscale image sensor or a color image sensor having an RGB color filter array deposited on its pixels). In another variation, the image acquisition assembly may include a three-chip, three-sensor (RGB) image sensor assembly  944 . 
     Processor and Controller 
     As shown in the schematic of  FIG. 6A , the system may include a processor  630 . The processor may include, for example, a microprocessor or other suitable central processing unit. In particular, the processor  630  may be configured to execute instructions to perform aspects of the methods described herein. As the low light video frames and/or the reference video frames are acquired, at least a portion of them may be stored in a memory unit for record-keeping purposes and/or retrieval for analysis during other aspects of the method, as described below. 
     As shown in the schematic of  FIG. 6A , the system may include a controller  640 , which may be embodied in, for example, a microprocessor and/or timing electronics. In some variations, a single image sensor may be used to acquire both low light video frames and reference video frames, and the controller  640  may control a timing scheme for the visible light source and/or the excitation light source, and the image acquisition assembly. This timing scheme may enable separation of the image signal associated with the low light signal and the image signal associated with the higher intensity reference light signal. In particular, the timing scheme may involve illuminating the object with illumination light and/or excitation light according to a pulsing scheme, and processing the low light image signal and reference image signal with a processing scheme, wherein the processing scheme is synchronized and matched to the pulsing scheme (e.g., via a controller) to enable separation of the two image signals in a time-division multiplexed manner. Examples of such pulsing and image processing schemes have been described in U.S. Pat. No. 9,173,554, filed on Mar. 18, 2009 and titled “IMAGING SYSTEM FOR COMBINED FULL-COLOR REFLECTANCE AND NEAR-INFRARED IMAGING,” the contents of which are incorporated in their entirety by this reference. However, other suitable pulsing and image processing schemes may be used to acquire reference video frames and low light video frames simultaneously, for example to acquire reflected light video frames and fluorescence video frames simultaneously. Furthermore, the controller may be configured to control the timing scheme for the visible light source and/or the excitation light source, and the image acquisition assembly based at least in part on the relative movement between the image acquisition assembly and the object. 
     Other Hardware 
     In some variations, the system may include image stabilizing technology that helps compensate for some ranges of motion (e.g., caused by unsteady hands holding the image acquisition assembly) in the acquired low light images and/or reference images. The image stabilizing technology may be implemented in hardware, such as with optical image stabilization technology that counteracts some relative movement between the image acquisition assembly and the object by varying the optical path to the image sensor (e.g., lens-based adjustments and/or sensor-based adjustments). Additionally, or alternatively, the image stabilization technology may be implemented in software, such as with digital image stabilization that counteracts some relative movement between the image acquisition assembly and the object (e.g., by shifting the electronic image between video frames, utilizing stabilization filters with pixel tracking, etc.). Such image stabilizing technology may, for example, help correct for motion blur in the characteristic low light video output (or in the acquired low light video frames) resulting from relative motion during long exposure periods or the combination of multiple low light video frames. 
     The system may, in some variations, include one or more hardware motion sensors (e.g., gyroscope, accelerometer) that measure absolute motion of the image acquisition assembly. Information from these motion-measuring sensors may be used, in addition or as an alternative to the above-described motion-estimation algorithms, to determine which imaging mode of the system is suitable for a given set of circumstances. 
     Additionally, the system may include one or more data modules  640  that communicates and/or stores some or all of the acquired reference video frames, acquired low light video frames, characteristic low light video output, and/or information generated from the image data. For instance, the data module  640  may include a display (e.g., computer screen or other monitor), recorder or other data storage device, printer, and/or picture archiving and communication system (PACS). The system may additionally or alternatively include any suitable systems for communicating and/or storing images and image-related data. 
     A kit may include any part of the systems described herein, and/or the tangible non-transitory computer-readable medium described above having computer-executable (readable) program code embedded thereon that may provide instructions for causing one or more processors, when executing the instructions, to perform one or more of the methods described herein. For instance, the instructions may cause one or more processors, when executing the instructions, to perform an adaptive imaging method for generating low light video of an object. The method comprises receiving a sequence of low light video frames depicting the object, wherein the low light video frames are acquired by an image acquisition assembly; assessing relative movement between the image acquisition assembly and the object based on reference video frames comprising at least a portion of the low light video frames and/or a portion of a substantially simultaneously acquired sequence of higher light intensity video frames; adjusting a level of image processing of the low light video frames based at least in part on the relative movement between the image acquisition assembly and the object; and generating a characteristic low light video output from a quantity of low light video frames, wherein the quantity of the low light video frames is based on the adjusted level of image processing of the low light video frames. Furthermore, the kit may include instructions for use of at least some of its components (e.g., for installing the computer-executable (readable) program code with instructions embedded thereon, etc.). 
     In other variations, a kit may include any part of the systems described herein and a fluorescence agent such as, for example, a fluorescence dye such as ICG or any suitable fluorescence agent or a combination of fluorescence agents. In some variations, a suitable fluorescence agent is an agent which can circulate with the blood (e.g., an agent which can circulate with, for example, a component of the blood such as plasma in the blood) and which fluoresces when exposed to appropriate excitation light energy. ICG, when administered to the subject, binds with blood proteins and circulates with the blood in the tissue. The fluorescence imaging agent (e.g., ICG) may be administered to the subject as a bolus injection (e.g., into a vein or an artery) in a concentration suitable for imaging such that the bolus circulates in the vasculature and traverses the microvasculature. In other embodiments in which multiple fluorescence imaging agents are used, such agents may be administered simultaneously, e.g. in a single bolus, or sequentially in separate boluses. In some embodiments, the fluorescence imaging agent may be administered by a catheter. In certain embodiments, the fluorescence imaging agent may be administered less than an hour in advance of performing the measurement of signal intensity arising from the fluorescence imaging agent. For example, the fluorescence imaging agent may be administered to the subject less than 30 minutes in advance of the measurement. In yet other embodiments, the fluorescence imaging agent may be administered at least 30 seconds in advance of performing the measurement. In still other embodiments, the fluorescence imaging agent may be administered contemporaneously with performing the measurement. According to some embodiments, the fluorescence imaging agent may be administered in various concentrations to achieve a desired circulating concentration in the blood. For example, in embodiments where the fluorescence imaging agent is ICG, it may be administered at a concentration of about 2.5 mg/mL to achieve a circulating concentration of about 5 μM to about 10 μM in blood. In various embodiments, the upper concentration limit for the administration of the fluorescence imaging agent is the concentration at which the fluorescence imaging agent becomes clinically toxic in circulating blood, and the lower concentration limit is the instrumental limit for acquiring the signal intensity data arising from the fluorescence imaging agent circulating with blood to detect the fluorescence imaging agent. In various other embodiments, the upper concentration limit for the administration of the fluorescence imaging agent is the concentration at which the fluorescence imaging agent becomes self-quenching. For example, the circulating concentration of ICG may range from about 2 μM to about 10 mM. Thus, in one aspect, the method comprises the step of administration of the imaging agent (e.g., a fluorescence imaging agent) to the subject and acquisition of the signal intensity data (e.g., video) prior to processing the signal intensity data according to the various embodiments. In another aspect, the method excludes any step of administering the imaging agent to the subject. 
     According to some embodiments, a suitable fluorescence imaging agent for use in fluorescence imaging applications to generate fluorescence image data is an imaging agent which can circulate with the blood (e.g., a fluorescence dye which can circulate with, for example, a component of the blood such as lipoproteins or serum plasma in the blood) and transit vasculature of the tissue (i.e., large vessels and microvasculature), and from which a signal intensity arises when the imaging agent is exposed to appropriate light energy (e.g., excitation light energy, or absorption light energy). In various embodiments, the fluorescence imaging agent comprises a fluorescence dye, an analogue thereof, a derivative thereof, or a combination of these. An example of the fluorescence agent is a fluorescence dye, which includes any non-toxic fluorescence dye. In certain variations, the fluorescence dye may include a dye that emits light in the near-infrared spectrum. In certain embodiments, the fluorescence dye may include a tricarbocyanine dye such as, for example, indocyanine green (ICG). In other variations, the fluorescence dye may comprise methylene blue, ICG or a combination thereof. In certain embodiments the dye is or comprises fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, rose Bengal, trypan blue, fluoro-gold, green fluorescence protein, flavins (e.g., riboflavin, etc.), methylene blue, porphysomes, cyanine dyes (e.g., cathepsin-activated Cy5 combined with a targeting ligand, Cy5.5, etc.), IRDye800CW, CLR 1502 combined with a targeting ligand, OTL38 combined with a targeting ligand, or a combination thereof, which is excitable using excitation light wavelengths appropriate to each imaging agent. In some variations, an analogue or a derivative of the fluorescence imaging agent may be used. For example, a fluorescence dye analogue or a derivative may include a fluorescence dye that has been chemically modified, but still retains its ability to fluoresce when exposed to light energy of an appropriate wavelength. In variations in which some or all of the fluorescence is derived from autofluorescence, one or more of the fluorophores giving rise to the autofluorescence may be an endogenous tissue fluorophore (e.g., collagen, elastin, NADH, etc.), 5-aminolevulinic acid (5-ALA), or a combination thereof. 
     In various embodiments, the fluorescence imaging agent may be provided as a lyophilized powder, solid, or liquid. In certain embodiments, the fluorescence imaging agent may be provided in a vial (e.g., a sterile vial), which may permit reconstitution to a suitable concentration by administering a sterile fluid with a sterile syringe. Reconstitution may be performed using any appropriate carrier or diluent. For example, the fluorescence imaging agent may be reconstituted with an aqueous diluent immediately before administration. In various embodiments, any diluent or carrier which will maintain the fluorescence imaging agent in solution may be used. As an example, ICG may be reconstituted with water. In some embodiments, once the fluorescence imaging agent is reconstituted, it may be mixed with additional diluents and carriers. In some embodiments, the fluorescence imaging agent may be conjugated to another molecule, such as a protein, a peptide, an amino acid, a synthetic polymer, or a sugar, for example to enhance solubility, stability, imaging properties, or a combination thereof. Additional buffering agents may optionally be added including Tris, HCl, NaOH, phosphate buffer, and/or HEPES. 
     A person of skill in the art will appreciate that, although a fluorescence imaging agent was described above in detail, other imaging agents may be used in connection with the systems, methods, and techniques described herein, depending on the medical imaging modality. 
     In some variations, the fluorescence imaging agent used in combination with the methods, systems and kits described herein may be used for blood flow imaging, tissue perfusion imaging, lymphatic imaging, or a combination thereof, which may be performed during an invasive surgical procedure, a minimally invasive surgical procedure, a non-invasive surgical procedure, or a combination thereof. Examples of invasive surgical procedure which may involve blood flow and tissue perfusion include a cardiac-related surgical procedure (e.g., CABG on pump or off pump) or a reconstructive surgical procedure. An example of a non-invasive or minimally invasive procedure includes wound (e.g., chronic wound such as for example pressure ulcers) treatment and/or management. In this regard, for example, a change in the wound over time, such as a change in wound dimensions (e.g., diameter, area), or a change in tissue perfusion in the wound and/or around the peri-wound, may be tracked over time with the application of the methods and systems. Examples of lymphatic imaging include identification of one or more lymph nodes, lymph node drainage, lymphatic mapping, or a combination thereof. In some variations, such lymphatic imaging may relate to the female reproductive system (e.g., uterus, cervix, vulva). 
     In variations relating to cardiac applications or any vascular applications, the imaging agent(s) (e.g., ICG alone or in combination with another imaging agent) may be injected intravenously. For example, the imaging agent may be injected intravenously through the central venous line, bypass pump and/or cardioplegia line and/or other vasculature to flow and/or perfuse the coronary vasculature, microvasculature and/or grafts. ICG may be administered as a dilute ICG/blood/saline solution down the grafted vessel or other vasculature such that the final concentration of ICG in the coronary artery or other vasculature depending on application is approximately the same or lower as would result from injection of about 2.5 mg (i.e., 1 ml of 2.5 mg/ml) into the central line or the bypass pump. The ICG may be prepared by dissolving, for example, 25 mg of the solid in 10 ml sterile aqueous solvent, which may be provided with the ICG by the manufacturer. One milliliter of the ICG solution may be mixed with 500 ml of sterile saline (e.g., by injecting 1 ml of ICG into a 500 ml bag of saline). Thirty milliliters of the dilute ICG/saline solution may be added to 10 ml of the subject&#39;s blood, which may be obtained in an aseptic manner from the central arterial line or the bypass pump. ICG in blood binds to plasma proteins and facilitates preventing leakage out of the blood vessels. Mixing of ICG with blood may be performed using standard sterile techniques within the sterile surgical field. Ten ml of the ICG/saline/blood mixture may be administered for each graft. Rather than administering ICG by injection through the wall of the graft using a needle, ICG may be administered by means of a syringe attached to the (open) proximal end of the graft. When the graft is harvested surgeons routinely attach an adaptor to the proximal end of the graft so that they can attach a saline filled syringe, seal off the distal end of the graft and inject saline down the graft, pressurizing the graft and thus assessing the integrity of the conduit (with respect to leaks, side branches etc.) prior to performing the first anastomosis. In other variations, the methods, dosages or a combination thereof as described herein in connection with cardiac imaging may be used in any vascular and/or tissue perfusion imaging applications. 
     Lymphatic mapping is an important part of effective surgical staging for cancers that spread through the lymphatic system (e.g., breast, gastric, gynecological cancers). Excision of multiple nodes from a particular node basin can lead to serious complications, including acute or chronic lymphedema, paresthesia, and/or seroma formation, when in fact, if the sentinel node is negative for metastasis, the surrounding nodes will most likely also be negative. Identification of the tumor draining lymph nodes (LN) has become an important step for staging cancers that spread through the lymphatic system in breast cancer surgery for example. LN mapping involves the use of dyes and/or radiotracers to identify the LNs either for biopsy or resection and subsequent pathological assessment for metastasis. The goal of lymphadenectomy at the time of surgical staging is to identify and remove the LNs that are at high risk for local spread of the cancer. Sentinel lymph node (SLN) mapping has emerged as an effective surgical strategy in the treatment of breast cancer. It is generally based on the concept that metastasis (spread of cancer to the axillary LNs), if present, should be located in the SLN, which is defined in the art as the first LN or group of nodes to which cancer cells are most likely to spread from a primary tumor. If the SLN is negative for metastasis, then the surrounding secondary and tertiary LN should also be negative. The primary benefit of SLN mapping is to reduce the number of subjects who receive traditional partial or complete lymphadenectomy and thus reduce the number of subjects who suffer from the associated morbidities such as lymphedema and lymphocysts. 
     The current standard of care for SLN mapping involves injection of a tracer that identifies the lymphatic drainage pathway from the primary tumor. The tracers used may be radioisotopes (e.g. Technetium-99 or Tc-99m) for intraoperative localization with a gamma probe. The radioactive tracer technique (known as scintigraphy) is limited to hospitals with access to radioisotopes require involvement of a nuclear physician and does not provide real-time visual guidance. A colored dye, isosulfan blue, has also been used, however this dye cannot be seen through skin and fatty tissue. In addition, blue staining results in tattooing of the breast lasting several months, skin necrosis can occur with subdermal injections, and allergic reactions with rare anaphylaxis have also been reported. Severe anaphylactic reactions have occurred after injection of isosulfan blue (approximately 2% of patients). Manifestations include respiratory distress, shock, angioedema, urticarial and pruritus. Reactions are more likely to occur in subjects with a history of bronchial asthma, or subjects with allergies or drug reactions to triphenylmethane dyes. Isosulfan blue is known to interfere with measurements of oxygen saturation by pulse oximetry and methemoglobin by gas analyzer. The use of isosulfan blue may result in transient or long-term (tattooing) blue coloration. 
     In contrast, fluorescence imaging in accordance with the various embodiments for use in SLN visualization, mapping, facilitates direct real-time visual identification of a LN and/or the afferent lymphatic channel intraoperatively, facilitates high-resolution optical guidance in real-time through skin and fatty tissue, visualization of blood flow, tissue perfusion or a combination thereof 
     In some variations, visualization, classification or both of lymph nodes during fluorescence imaging may be based on imaging of one or more imaging agents, which may be further based on visualization and/or classification with a gamma probe (e.g., Technetium Tc-99 m  is a clear, colorless aqueous solution and is typically injected into the periareolar area as per standard care), another conventionally used colored imaging agent (isosulfan blue), and/or other assessment such as, for example, histology. The breast of a subject may be injected, for example, twice with about 1% isosulfan blue (for comparison purposes) and twice with an ICG solution having a concentration of about 2.5 mg/ml. The injection of isosulfan blue may precede the injection of ICG or vice versa. For example, using a TB syringe and a 30 G needle, the subject under anesthesia may be injected with 0.4 ml (0.2 ml at each site) of isosulfan blue in the periareolar area of the breast. For the right breast, the subject may be injected at 12 and 9 o&#39;clock positions and for the left breast at 12 and 3 o&#39;clock positions. The total dose of intradermal injection of isosulfan blue into each breast may be about 4.0 mg (0.4 ml of 1% solution: 10 mg/ml). In another exemplary variation, the subject may receive an ICG injection first followed by isosulfan blue (for comparison). One 25 mg vial of ICG may be reconstituted with 10 ml sterile water for injection to yield a 2.5 mg/ml solution immediately prior to ICGadministration. Using a TB syringe and a 30G needle, for example, the subject may be injected with about 0.1 ml of ICG (0.05 ml at each site) in the periareolar area of the breast (for the right breast, the injection may be performed at 12 and 9 o&#39;clock positions and for the left breast at 12 and 3 o&#39;clock positions). The total dose of intradermal injection of ICG into each breast may be about 0.25 mg (0.1 ml of 2.5 mg/ml solution) per breast. ICG may be injected, for example, at a rate of 5 to 10 seconds per injection. When ICG is injected intradermally, the protein binding properties of ICG cause it to be rapidly taken up by the lymph and moved through the conducting vessels to the LN. In some variations, the ICG may be provided in the form of a sterile lyophilized powder containing 25 mg ICG with no more than 5% sodium iodide. The ICG may be packaged with aqueous solvent consisting of sterile water for injection, which is used to reconstitute the ICG. In some variations the ICG dose (mg) in breast cancer sentinel lymphatic mapping may range from about 0.5 mg to about 10 mg depending on the route of administration. In some variations, the ICG does may be about 0.6 mg to about 0.75 mg, about 0.75 mg to about 5 mg, about 5 mg to about 10 mg. The route of administration may be for example subdermal, intradermal (e.g., into the periareolar region), subareolar, skin overlaying the tumor, intradermal in the areola closest to tumor, subdermal into areola, intradermal above the tumor, periareolar over the whole breast, or a combination thereof. The NIR fluorescent positive LNs (e.g., using ICG) may be represented as a black and white NIR fluorescence image(s) for example and/or as a full or partial color (white light) image, full or partial desaturated white light image, an enhanced colored image, an overlay (e.g., fluorescence with any other image), a composite image (e.g., fluorescence incorporated into another image) which may have various colors, various levels of desaturation or various ranges of a color to highlight/visualize certain features of interest. Processing of the images may be further performed for further visualization and/or other analysis (e.g., quantification). The lymph nodes and lymphatic vessels may be visualized (e.g., intraoperatively, in real time) using fluorescence imaging systems and methods according to the various embodiments for ICG and SLNs alone or in combination with a gamma probe (Tc-99m) according to American Society of Breast Surgeons (ASBrS) practice guidelines for SLN biopsy in breast cancer patients. Fluorescence imaging for LNs may begin from the site of injection by tracing the lymphatic channels leading to the LNs in the axilla. Once the visual images of LNs are identified, LN mapping and identification of LNs may be done through incised skin, LN mapping may be performed until ICG visualized nodes are identified. For comparison, mapping with isosulfan blue may be performed until ‘blue’ nodes are identified. LNs identified with ICG alone or in combination with another imaging technique (e.g., isosulfan blue, and/or Tc-99m) may be labeled to be excised. Subject may have various stages of breast cancer (e.g., IA, IB, IIA). 
     In some variations, such as for example, in gynecological cancers (e.g., uterine, endometrial, vulvar and cervical malignancies), ICG may be administered interstitially for the visualization of lymph nodes, lymphatic channels, or a combination thereof. When injected interstitially, the protein binding properties of ICG cause it to be rapidly taken up by the lymph and moved through the conducting vessels to the SLN. ICG may be provided for injection in the form of a sterile lyophilized powder containing 25 mg ICG (e.g., 25 mg/vial) with no more than 5.0% sodium iodide. ICG may be then reconstituted with commercially available water (sterile) for injection prior to use. According to an embodiment, a vial containing 25 mg ICG may be reconstituted in 20 ml of water for injection, resulting in a 1.25 mg/ml solution. A total of 4 ml of this 1.25 mg/ml solution is to be injected into a subject (4×1 ml injections) for a total dose of ICG of 5 mg per subject. The cervix may also be injected four (4) times with a 1 ml solution of 1% isosulfan blue 10 mg/ml (for comparison purposes) for a total dose of 40 mg. The injection may be performed while the subject is under anesthesia in the operating room. In some variations the ICG dose (mg) in gynecological cancer sentinel lymph node detection and/or mapping may range from about 0.1 mg to about 5 mg depending on the route of administration. In some variations, the ICG does may be about 0.1 mg to about 0.75 mg, about 0.75 mg to about 1.5 mg, about 1.5 mg to about 2.5 mg, about 2.5 mg to about 5 mg. The route of administration may be for example cervical injection, vulva peritumoral injection, hysteroscopic endometrial injection, or a combination thereof. In order to minimize the spillage of isosulfan blue or ICG interfering with the mapping procedure when LNs are to be excised, mapping may be performed on a hemi-pelvis, and mapping with both isosulfan blue and ICG may be performed prior to the excision of any LNs. LN mapping for Clinical Stage I endometrial cancer may be performed according to the NCCN Guidelines for Uterine Neoplasms, SLN Algorithm for Surgical Staging of Endometrial Cancer; and SLN mapping for Clinical Stage I cervical cancer may be performed according to the NCCN Guidelines for Cervical Neoplasms, Surgical/SLN Mapping Algorithm for Early-Stage Cervical Cancer. Identification of LNs may thus be based on ICG fluorescence imaging alone or in combination or co-administration with for a colorimetric dye (isosulfan blue) and/or radiotracer. 
     Visualization of lymph nodes may be qualitative and/or quantitative. Such visualization may comprise, for example, lymph node detection, detection rate, anatomic distribution of lymph nodes. Visualization of lymph nodes according to the various embodiments may be used alone or in combination with other variables (e.g., vital signs, height, weight, demographics, surgical predictive factors, relevant medical history and underlying conditions, histological visualization and/or assessment, Tc-99m visualization and/or assessment, concomitant medications). Follow-up visits may occur on the date of discharge, and subsequent dates (e.g., one month). 
     Lymph fluid comprises high levels of protein, thus ICG can bind to endogenous proteins when entering the lymphatic system. Fluorescence imaging (e.g., ICG imaging) for lymphatic mapping when used in accordance with the methods and systems described herein offers the following example advantages: high-signal to background ratio (or tumor to background ratio) as NIR does not generate significant autofluorescence, real-time visualization feature for lymphatic mapping, tissue definition (i.e., structural visualization), rapid excretion and elimination after entering the vascular system, and avoidance of non-ionizing radiation. Furthermore, NIR imaging has superior tissue penetration (approximately 5 to 10 millimeters of tissue) to that of visible light (1 to 3 mm of tissue). The use of ICG for example also facilitates visualization through the peritoneum overlying the para-aortic nodes. Although tissue fluorescence can be observed with NIR light for extended periods, it cannot be seen with visible light and consequently does not impact pathologic evaluation or processing of the LN. Also, florescence is easier to detect intra-operatively than blue staining (isosulfan blue) of lymph nodes. In other variations, the methods, dosages or a combination thereof as described herein in connection with lymphatic imaging may be used in any vascular and/or tissue perfusion imaging applications. 
     Tissue perfusion relates to the microcirculatory flow of blood per unit tissue volume in which oxygen and nutrients are provided to and waste is removed from the capillary bed of the tissue being perfused. Tissue perfusion is a phenomenon related to but also distinct from blood flow in vessels. Quantified blood flow through blood vessels may be expressed in terms that define flow (i.e., volume/time), or that define speed (i.e., distance/time). Tissue blood perfusion defines movement of blood through micro-vasculature, such as arterioles, capillaries, or venules, within a tissue volume. Quantified tissue blood perfusion may be expressed in terms of blood flow through tissue volume, namely, that of blood volume/time/tissue volume (or tissue mass). Perfusion is associated with nutritive blood vessels (e.g., micro-vessels known as capillaries) that comprise the vessels associated with exchange of metabolites between blood and tissue, rather than larger-diameter non-nutritive vessels. In some embodiments, quantification of a target tissue may include calculating or determining a parameter or an amount related to the target tissue, such as a rate, size volume, time, distance/time, and/or volume/time, and/or an amount of change as it relates to any one or more of the preceding parameters or amounts. However, compared to blood movement through the larger diameter blood vessels, blood movement through individual capillaries can be highly erratic, principally due to vasomotion, wherein spontaneous oscillation in blood vessel tone manifests as pulsation in erythrocyte movement. 
     One or more embodiments are directed to a fluorescence imaging agent for use in the imaging systems and methods as described herein. In one or more embodiments, the use may comprise blood flow imaging, tissue perfusion imaging, lymphatic imaging, or a combination thereof, which may occur during an invasive surgical procedure, a minimally invasive surgical procedure, a non-invasive surgical procedure, or a combination thereof. The fluorescence agent may be included in the kit described herein. 
     In one or more embodiments, the invasive surgical procedure may comprise a cardiac-related surgical procedure or a reconstructive surgical procedure. The cardiac-related surgical procedure may comprise a cardiac coronary artery bypass graft (CABG) procedure which may be on pump and/or off pump. 
     In one or more embodiments, the minimally invasive or the non-invasive surgical procedure may comprise a wound care procedure. 
     In one or more embodiments, the lymphatic imaging may comprise identification of a lymph node, lymph node drainage, lymphatic mapping, or a combination thereof. The lymphatic imaging may relate to the female reproductive system. 
     The methods and processes described herein may be performed by code or instructions to be executed by a computer, processor, manager, or controller, or in hardware or other circuitry. Because the algorithms that form the basis of the methods (or operations of the computer, processor, or controller) are described in detail, the code or instructions for implementing the operations of the method embodiments may transform the computer, processor, or controller into a special-purpose processor for performing the methods described herein. 
     Also, another embodiment may include a computer-readable medium, e.g., a non-transitory computer-readable medium, for storing the code or instructions described above. The computer-readable medium may be a volatile or non-volatile memory or other storage device, which may be removably or fixedly coupled to the computer, processor, or controller which is to execute the code or instructions for performing the method embodiments described herein. 
     Example embodiments have been disclosed herein, and although specific terms are employed, they are used and are to be interpreted in a generic and descriptive sense only and not for purpose of limitation. In some instances, as would be apparent to one of ordinary skill in the art as of the filing of the present application, features, characteristics, and/or elements described in connection with a particular embodiment may be used singly or in combination with features, characteristics, and/or elements described in connection with other embodiments unless otherwise specifically indicated. Accordingly, it will be understood by those of skill in the art that various changes in form and details may be made without departing from the spirit and scope of the present invention as set forth in the following. 
     While the present disclosure has been illustrated and described in connection with various embodiments shown and described in detail, it is not intended to be limited to the details shown, since various modifications and structural changes may be made without departing in any way from the scope of the present disclosure. Various modifications of form, arrangement of components, steps, details and order of operations of the embodiments illustrated, as well as other embodiments of the disclosure may be made without departing in any way from the scope of the present disclosure, and will be apparent to a person of skill in the art upon reference to this description. It is therefore contemplated that the appended claims will cover such modifications and embodiments as they fall within the true scope of the disclosure. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the disclosure includes embodiments having combinations of all or some of the features described. For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.