Visualization technologies for 5-ALA-based fluorescence-guided surgeries

Abstract

5-ALA-based fluorescence-guided surgery has been shown to be a safe and effective method to improve intraoperative visualization and resection of malignant gliomas. However, it remains ineffective in guiding the resection of lower-grade, non-enhancing, and deep-seated tumors, mainly because these tumors do not produce detectable fluorescence with conventional visualization technologies, namely, wide-field (WF) surgical microscopy. Here, we describe some of the main factors that limit the sensitivity and accuracy of conventional WF surgical microscopy, and then provide a survey of commercial and research prototypes being developed to address these challenges. The basic principles of these technologies are described, along with their advantages and disadvantages, as well as the current tatus of clinical translation for each technology. Within this technical review, a goal is to also provide a neurosurgical perspective on how these visualization technologies might best be implemented for guiding glioma surgeries in the future.

Introduction

Cytoreduction remains the first step in effective glioma management, in which the primary objective is to maximize the extent of resection while avoiding neurological damage [1,2]. Glioma margins, however, are ambiguous under gross visual inspection and with standard low-power white-light surgical microscopes [3,4]. One approach to improve the visualization of bulk tumor regions is to use tumor fluorescence to highlight abnormal cells and improve intraoperative visual contrast with respect to the surrounding normal brain tissues. In recent decades, only a few fluorescence contrast agents, such as indocyanine green (ICG) and sodium fluorescein, have been available for intraoperative use in humans. However, in 2017, the United States Food and Drug Administration (U.S. FDA) approved the first metabolic (i.e. targeted) optical imaging agent, 5-aminolevulinic acid (5-ALA) (commercially known as Gliolan®), for use during glioma surgeries to enhance the visualization of malignant tissue [5]. In brief, 5-ALA is a non-fluorescent prodrug that induces the preferential accumulation of a fluorescent byproduct, protoporphyrin IX (PpIX), in proliferating tumor cells. It has been shown that the level of PpIX in gliomas correlates with tumor cell density and a number of proliferative biomarkers such as Ki-67, mitochondrial activity, etc [6–10]. It has been shown that 5-ALA-based fluorescence-guided surgeries (FGS) can significantly improve the rate of gross total resections (based on post-operative MRI) of high grade gliomas (HGG) in a collection of clinical studies worldwide [8,10–21], including a landmark randomized controlled phase III trial in Germany [22]. While this visualization method is simple and is effective in detecting strong fluorescence from HGG, it is less effective for identifying residual tumor infiltration beyond MRI contrast-enhancing regions, where the fluorescence signal declines (presumably due to a reduction of tumor cell density and PpIX levels). This method is also less effective for guiding the resection of low grade gliomas (LGGs), which often do not generate sufficient PpIX fluorescence to enable detection by conventional visualization technologies. Fortunately, it has been shown that with more advanced visualization technologies, intraoperative detection of PpIX fluorescence in LGGs [23,24], at infiltrative margins [9,25,26], and from sub-surface tumors [27–33] is possible, potentially extending the utility of 5-ALA-based FGS. Here, we survey the current commercial products and research prototypes being developed for visualizing 5-ALA-induced fluorescence in gliomas. The basic principles of these technologies will be described, along with their advantages and disadvantages and the current status of clinical translation for each technology. Finally, a neurosurgical perspective will be provided on how these visualization technologies can best be implemented for guiding glioma surgeries in the future.

Visualization technologies

Visualization technologies for 5-ALA-induced fluorescence in gliomas can be grouped into free-standing systems and probe-based systems (Fig. 1). Free-standing imaging systems consist of standard wide-field (WF) operating microscopes (e.g. Zeiss® Pentero, Leica® M530, etc.) or other custom microscopes that operate on a similar principle: namely, by illuminating a large field (usually the entire surgical cavity) at one excitation wavelength while imaging the resultant fluorescence signal onto a detector array (camera), or through an eyepiece, to the unaided eyes of the surgeon. Such techniques have the advantage of providing a large field-of-view (FOV), but are incapable of visualizing individual cells due to a trade-off between FOV and spatial resolution. Since each pixel of a low-resolution WF image represents an average signal from a large number of cells, all of which can produce non-specific autofluorescence and other background fluorescence, the sensitivity to detect sparse fluorescently labeled cell populations (e.g. disseminated tumor cells at the margins) is reduced (Fig. 2a). In addition, the long working distance of free-standing surgical microscopes results in a further reduction in optical detection sensitivity (inverse-square-law). In contrast to free-standing microscopes, probe-based imaging systems are designed to provide a higher-magnification view of localized regions within the surgical site, with FOVs ranging from sub-millimeter to centimeter scales. Probe-based methods in general imply higher sensitivity because the probes are designed to be placed very close to (a few millimeters at most) or in direct contact with the tissue surface, resulting in more efficient light delivery and collection compared to non-contact visualization methods [3] (Fig. 2b). In the following sections, we provide a brief discussion of the strengths and weaknesses of the current technologies for 5-ALA-based FGS in gliomas as a starting point for comparisons with other visualization systems that are under development for PpIX-guided neurosurgical resection.

Fig. 1.

Comparison of common visualization technologies for 5-ALA-based FGS of gliomas. (a) Imaging methods based on free-standing microscopes typically provide a large FOV that can often visualize the entire surgical cavity. (b) Example image from a surgical fluorescence wide-field microscope. The pink fluorescing region corresponds to the bulk tumor while the blue back-reflected light is from the illumination source at ~405 nm. (c) Alternatively, the signal can be spectrally resolved into multiple wavelength channels, which can be analyzed using mathematical models to correct for nonlinear tissue optical properties, which provides more accurate quantification of fluorophore concentrations. In this example [25], a quantitative spectrally resolved image is overlaid on a white-light image of the surgical cavity. (d) Probe-based methods are designed to be placed very close to, or in direct contact with the tissue surface, providing a number of advantages such as flexibility to access difficult-to-reach locations within the tumor cavity, and high optical-detection sensitivity, at the trade-off of a reduced FOV. (e) Example fluorescence image from a high-resolution optical-sectioning microscope showing fluorescence expression at the microscopic (sub-cellular) scale. (f) Example data generated by a spectroscopy probe. In contrast to the spectral imaging data shown in (c), the data shown in (f) exhibits higher optical detection sensitivity but lacks spatial information (non-imaging).

Fig. 2.

Factors that affect the sensitivity of tumor detection. (a) The “averaging effect” due to low spatial resolution reduces the signal-to-background ratio of optical detection, which therefore reduces the ability to detect sparse disseminated cells that exhibit a differentially higher level of fluorescence compared to adjacent benign cells. (b) A longer working distance is typically associated with reduced sensitivity due to an inverse-square-law reduction in signal collection with distance. Example images of PpIX fluorescence using a wide-field (WF) scanning fiber endoscope at different distances from the tumor (Ref. [9]). (c) Wide-field imaging in thick tissue is subject to a strong background due to insufficient rejection of out-of-focus and multiply scattered light. High-resolution optical-sectioning techniques, such as confocal microscopy, can enable sensitive detection of individually labeled cells and sub-cellular features. Example images of PpIX fluorescence of a human glioma biopsy using WF microscopy, which generates images with lower contrast, and confocal microscopy, which generates images with higher contrast (Ref. [40]).

Wide-field surgical microscopy

The first report on 5-ALA-based FGS of gliomas was published in 1998 by Dr. Walter Stummer and colleagues in Germany [34,35]. The benefits of this new method were subsequently demonstrated and documented in a collection of reports from around the world, which prompted the development of commercial wide-field fluorescence microscopy systems (e.g. Zeiss® Blue400 and Leica® FL400) for 5-ALA-based FGS. These systems consist of add-on modules to existing white-light surgical microscopes (e.g. Zeiss® Pentero and Leica® M530), with optimized filter sets for exciting and detecting PpIX fluorescence. For example, the Zeiss Blue400 module consists of a filter set that provides excitation at 400–410 nm and collection at 620–710 nm, both of which are optimally matched to the excitation and emission spectra of PpIX. In comparison to the early systems used by Stummer et al. in 1998 (Ex @ 375–440 nm; Em @ >455 nm), the newer systems provide higher excitation efficiency as well as superior rejection of autofluorescence background and back-scattered excitation light.

As a commercialized technology for 5-ALA-based FGS, free-standing WF surgical microscopy provides a straightforward and intuitive means to visualize macroscopic PpIX fluorescence in real time over a large FOV. However, as previously mentioned within this article and other reports [3,36–39], this method has certain limitations. First, WF imaging often does not provide sufficient resolution and sensitivity to visualize sparse foci of fluorescence, such as the weak sub-cellular distribution of PpIX in LGGs and disseminated tumor cells at the diffuse margins of all gliomas. Second, WF imaging in thick tissue is subject to large levels of background signal due to the inefficient rejection of out-of-focus light, further reducing its sensitivity to detect weak fluorescence (Fig. 2c). Third, there is often a steep angle of incidence between the focal plane of standard operating microscopes and the walls of a resection cavity, which results in defocused regions and blind spots, especially within sulci and behind bends. Finally, almost all current WF techniques for visualizing of PpIX fluorescence are not quantitative but rather rely upon the subjective judgements of the surgeon to determine what colors and intensities correspond to tumor, benign, and/or mixed cell populations. In short, since the detected fluorescence signal is influenced by a number of parameters such as tissue optical properties (e.g. scattering and absorption) and non-specific auto-fluorescence background, there is a nonlinear and often non-intuitive relationship between the detected fluorescence signal and actual fluorophore concentrations.

Wide-field fluorescence endoscopy

Initial efforts to develop endoscopic probes for 5-ALA-based FGS aimed to provide visualization of PpIX fluorescence from deep-seated tumors and other regions that are not easily accessed by standard free-standing surgical microscopes. In general, the detection sensitivity of such endoscopic imaging probes is superior to that of conventional surgical microscopes due to the shorter working distance of the miniature probes that are positioned much closer to the tissue surface, but at the cost of a reduced FOV. In 2007, Tamura and colleagues reported on the use of a modified commercial endoscope to visualize PpIX fluorescence in an intraventricular malignant glioma and used this method to obtain an image-guided biopsy that was confirmed malignant by histopathology [41]. As noted by the authors, the main modifications made to the endoscopic system, to enable PpIX visualization, were the use of a high power (300 mW) 405-nm laser source in addition to a high sensitivity CCD camera. This study did not report the effect of photo-bleaching, but it should be noted that the illumination power used was significantly higher than in other studies in which photobleaching was observed [9,42]. At around the same time, Potapov and colleagues also performed a pilot study using a custom endoscope (now commercially available) to visualize PpIX fluorescence in 17 patients (mostly grade IV gliomas) [43]. According to the surgeons involved in the study, the endoscope allowed for more-thorough inspection of the surgical cavity without applying traction to the brain tissue (the practice of using surgical tools to reveal blind spots, which can potentially be harmful to the patient). More recently, Belykh and colleagues showed that the use of a scanning fiber endoscope (SFE) enabled the detection of PpIX fluorescence near the margin of an infiltrative glioma in a preclinical mouse model for which the fluorescence level was below the detection limit of a standard wide-field operating microscope [9]. The SFE technology utilizes an innovative scanning mechanism to rapidly actuate a single-mode fiber tip, allowing for high-resolution imaging (spatial resolution of ~20 μm) at video rate with an extremely compact probe head (Ø ~2 mm).

Quantitative spectroscopy

As previously mentioned, the raw intensity detected by a fluorescence microscope can be misleading because it is affected by tissue-dependent optical properties such as light absorption and scattering, as well as optical detection parameters such as working distance and angle of detection. Unlike fluorescence microscopy, which measures the total signal within a relatively broad range of wavelengths (tens to hundreds of nanometers), spectroscopy measures the signal within individual wavelength bins over a wide spectrum. Studies have shown that this spectrally resolved fluorescence signal can be used to infer certain tissue optical properties with the aid of numerical models, resulting in more accurate and sensitive measurements of the relative, or in some implementations, absolute concentration of fluorophores such as PpIX. A number of different spectroscopic probes have been developed by research groups and companies for 5-ALA-based FGS of gliomas [8,44–47], and have also been demonstrated in a collection of clinical studies to improve the sensitivity of PpIX fluorescence detection in patients with LGGs [8,10,24]. In addition to providing more accurate and linear measurements of PpIX concentration, these spectroscopic probes share the advantages of other probe-based imaging modalities such as flexibility and improved sensitivity compared to free-standing imaging methods. The basic design principles for many of the spectroscopic probes that have been developed for biomedical applications have been outlined in a previous review article [48].

As mentioned in the previous paragraph, probe-based spectroscopy can provide accurate quantification of PpIX concentration at localized points of interests. However, it is usually impractical to achieve PpIX quantification across an entire surgical cavity. In 2012, Valdes and colleagues developed a WF imaging version of intraoperative spectroscopy to quantify PpIX concentrations over a large and spatially resolved FOV (i.e. a PpIX map) at low magnification [25,49]. This system was based on a standard surgical microscope (Zeiss Pentero), in which the main modification was the placement of a liquid crystal tunable filter in front of a monochrome digital camera to achieve spectrally resolved detection over time. A trade off is that the frame rate was limited to ~ 1 Hz to achieve sufficient spectral resolution and signal-to-noise ratio for a spectrally resolved image over a large area [25,49]. More recently, refined algorithms have been developed to process these hyperspectral images, in which improved sensitivity and accuracy of PpIX quantification has been demonstrated in image phantoms and in patients [50].

Deep-tissue imaging techniques

Deep tissue imaging is desirable to detect tumor infiltrates and residual tumors below the surgical margin, but visualizing PpIX fluorescence at depth is challenging due to the fact that PpIX is optimally excited at 405 nm, a wavelength that is subject to strong scattering in tissue as well as strong absorption by blood [51]. If high-resolution (cellular resolution or better) imaging is desired, imaging depths are typically limited to ~100 microns in tissue. However, PpIX exhibits a broad excitation spectrum with a few minor (weaker) absorption peaks at longer wavelengths, which allows for larger penetration depths at the cost of reduced efficiency of fluorescence generation. A number of fluorescence tomography systems have been designed to operate near 630 nm and have been shown to successfully detect subsurface PpIX fluorescence at depths of up to a few millimeters in tissue [27,33,31], including intact mouse brain [28]. These tomographic systems typically have a limited frame rate due to the long integration time (on the order of a minute) that is necessary to achieve sufficient signal-to-noise ratio, in addition to the time-consuming process of performing tomographic reconstruction. A major difference amongst various systems has been the illumination/detection method employed. For example, in 2009, Kepshire and colleagues developed a system that enabled tomographic PpIX imaging through an intact mouse skull, in which the illumination beams were launched at multiple angles using fan-beam scanners, and detection was performed with an array of time-resolved photomultiplier tubes (PMT) [28], a setup that is similar to standard X-ray-based CT. In 2012, Konecky and colleagues developed a fluorescence tomography system based on spatial frequency domain imaging (SFDI) [27], a relatively new technique that uses spatially modulated (i.e. patterned) illumination light to achieve quantitative depth-resolved imaging [52]. This system also utilized multiple wavelengths to correct for changes in tissue optical properties, and to therefore achieve more-accurate quantification of PpIX concentrations. Other model-based methods have also been explored to quantify fluorophores at depth by exploiting the redshift of the emission spectrum due to depth-dependent attenuation of light [30–32,53]. Based on the theory in Ref. 30, wide-field imaging systems have been developed with red-light excitation to provide depth-resolved and quantitative measurement of PpIX up to several millimeters deep in turbid media with spatial resolution of about 1 mm [31,32]. The clinical feasibility of this technique has also been demonstrated recently by Roberts and colleagues in a clinical study of 29 patients (including HGG, LGG, and other brain tumors), in which PpIX fluorescence was successfully detected at depths up to 5 mm using red-light (620–640 nm) illumination and detection through a 650-nm long-pass filter [33] (Fig. 3).

Fig. 3.

Example images of WF imaging methods. (a) The cortical surface under white-light illumination. (b) For patients administered with 5-ALA prior to surgery, blue-light illumination is used to generate red fluorescence (from 5-ALA-induced PpIX) from bulk tumor regions. (c) Under red-light illumination, deeper light penetration into tissue is achieved, which reveals PpIX contributions from both superficial and subsurface tumor (represented with a false colormap). (d) Reconstruction of MRI data showing a weighted average of the first 1 mm below the cortical surface. (e) Reconstruction of MRI data showing a weighted average of the first 5 mm below the cortical surface. It should be noted that the fluorescence image generated with blue-light excitation (panel b) more-closely matches the MRI data integrated over a 1-mm depth (panel d) whereas the fluorescence image generated with red-light excitation (panel c) more closely matches the MRI data integrated over a 5-mm depth (panel e). Figure reprinted from Ref. [33].

Probe-based confocal microscopy

Confocal microscopy is perhaps the most ubiquitous optical-sectioning microscopy technique, in which spatial filters (such as pinholes and slits) are used to reject out-of-focus and multiply scattered background light. Confocal microscopes can generate high image contrast and micron-scale spatial resolution at imaging depths of up to ~100 microns within thick tissues. Wei, Sanai, Liu and colleagues recently showed that quantification of PpIX expression with ex vivo confocal microscopy of thick brain specimens correlated well with quantification of PpIX via slide-based fluorescence histology [54]. In terms of in vivo imaging, in 2011, Sanai and colleagues reported on the use of a handheld confocal microscope probe to visualize microscopic PpIX expression in LGGs within patients, in which PpIX fluorescence was undetectable with low-magnification WF fluorescence surgical microscopy [23]. It should be noted that the commercial intraoperative confocal microscope utilized in the pilot study was not optimized for visualizing PpIX fluorescence since an excitation wavelength of 488 nm was utilized instead of a more-optimal wavelength of 405 nm. Furthermore, this device, which was based on a conventional single-axis confocal architecture, had a relatively slow frame rate of < 1 Hz that was limited by the miniature 2D scanning mechanism used in the device, which made it susceptible to motion artifacts (blurring and image distortion) during handheld use. In order to overcome some of the limitations of previous confocal microscopy probes, a handheld line-scanned dual-axis confocal microscope has been developed [55] and continues to be refined for high-speed visualization of PpIX fluorescence in gliomas. In brief, this new device utilizes a dual-axis confocal architecture (Fig. 4) with spatially separated illumination and collection beams to achieve high image contrast – due to more efficient rejection of out-of-focus and multiply scattered light in comparison to conventional single-axis confocal microscope – with the added advantage that chromatic differences between the beams are less problematic since the illumination and collection beams can be independently aligned. The device utilizes a line-by-line scanning mechanism that enables a high frame rate of >15 Hz, which minimizes motion artifacts during handheld use. Technical details about this device are contained in a recent report and review [55,56]. A general overview of various miniature in vivo microscopy techniques may also be found in a separate review article [57].

Fig. 4.

Comparison of common contact probe designs for PpIX visualization. (a) Spectroscopy probes collect signal from a large region defined by the illumination (blue) and collection (red) beams, and typically do not provide spatial information (i.e. non-imaging). In practice, most probes use multiple collection fibers or angled fibers to increase the optical detection sensitivity. (b) A conventional confocal microscope uses a single high-NA objective lens for both illumination and collection. (c) Dual-axis confocal microscopy utilizes off-axis and spatially separated low-NA illumination and collection beams, which provides superior image contrast (signal to background ratio), certain advantages for miniaturization, and comparable spatial resolution to conventional single-axis confocal microscopes.

A number of intraoperative confocal probes have also been developed based on coherent fiber bundles [58,59], which eliminate the need to incorporate a complex scanning mechanism in the probe head and thus provides advantages such as high flexibility, small form factors, and high frame rates. A commercial fiber-bundle confocal endomicroscope developed by Mauna Kea Technologies (MKT) has recently received FDA approval for neurosurgical use, with separate devices designed to operate at 488 nm and 800 nm, which are the optimal wavelengths to visualize fluorescein and indocyanine green, respectively. The major limitation of this technology for visualizing PpIX fluorescence is that the ion-doped fiber bundles in these systems generate significant auto-fluorescence in the 600–700 nm wavelength range when the fibers are used to transmit 405-nm illumination light, which overwhelms the PpIX signal collected from biological tissues [60]. In addition, the MKT device does not allow for adjustment of the imaging depth for any single fiber-bundle probe.

Summary & discussion

Free-standing, non-quantitative WF microscopy remains the most widely used visualization technology for 5-ALA-based FGS of gliomas, and has been shown to be a clinically safe and simple method to enhance the intraoperative visualization of malignant gliomas compared to conventional methods. However, this method still lacks the sensitivity to effectively guide the resection of tumor in LGGs, or at the infiltrative margins of all gliomas, where tumor burden decreases. As summarized in Fig. 2, some of the key factors that limit the sensitivity of free-standing WF microscopy are: 1) the “averaging effect” due to low spatial resolution, which reduces the contrast for detecting disseminated and sparse cells; 2) the poor optical detection sensitivity due to the long WD of a free-standing microscope; and 3) the higher background due to out-of-focus and scattered light, in comparison to optical-sectioning techniques that suppress this tissue-induced optical background. Furthermore, it is necessary to maintain a constant WD and a perpendicular angle between the WF microscope and the tissue in order to generate high-quality images. Tumor cavities are often out-of-range or not completely in focus for the fixed-WD fluorescence microscopes used for FGS. In addition, sidewalls are difficult to visualize because of their steep angle with respect to the illumination source and optics. WF systems that are capable of quantitative and subsurface fluorophore imaging have been developed but are not yet commercially available. A number of WF fluorescence tomography systems have been developed to detect deep-seated tumors below the surgical surface, but have typically improved the imaging depth at the cost of further reducing optical detection sensitivity. Probe-based imaging methods have been developed to mitigate many of the limitations of WF imaging systems, aiming to provide more sensitive and quantitative detection of PpIX fluorescence in gliomas, at the cost of a smaller FOV and increased design complexity. The key trade-offs and the status of clinical translation of these imaging systems have been summarized in Table 1. A partial list of clinical studies that are associated with these imaging technologies have been listed in Table 2.

Table 1.

Summary of key specifications and trade-offs

Technology	Free-standing			Probe-based system
	Wide-field microscopy	Wide-field spectroscopy	Fluorecence tomography	Wide-filed endoscopy	Fiber-optic spectroscopy	Point-scanned confocal	Line-scanned confocal
Field of view	☆☆☆☆	☆☆☆☆	☆☆☆☆	☆☆	☆☆	☆	☆
Spatial resolution	☆☆	☆☆	☆	☆☆☆	☆	☆☆☆☆	☆☆☆☆
Imaging depth	☆	☆	☆☆☆☆	☆	☆☆☆	☆☆	☆☆
Frame rate	☆☆☆☆	☆☆	☆	☆☆☆☆	☆☆☆	☆☆	☆☆☆☆
Sensitivity	☆	☆	☆	☆☆☆	☆☆☆	☆☆☆☆	☆☆☆☆
Direct contact with patients	No	No	No	No	Yes	Yes	Yes
Commercially avaliable	Yes	No	No	Yes	No	Yes	No
Related clinical studies	11–18, 22	25, 49, 50	33	9, 10, 24	8, 10, 24	23	N/A

Table 2.

Summary of clinical studies

Year & Authors	Visualization technology	# of patients	Grade of gliomas	GTR%	Specificity	Sensitivity
2000, Stummer et al.	WF surgical microsopy	52	HGG	63%	-	-
2000, Stummer et al.	WF surgical microsopy	270	HGG	65%	-	-
2011, Roberts et al.	WF surgical microsopy	11	HGG	-	71%	75%
2011,valdes et al.	Probe-based spectroscopy	14	HGG & LGG	-	92%	84%
2014, Stummer et al.	Probe-based spectroscopy	13	HGG	-	95% & 41%*	72% & 94%*
2014,Belloch et al.	Probe-based WF microscopy	21	HGG	71%	-	-
2041, Rapp et al.	Probe-based WF microscopy	8	HGG	-	87%	100%
2015,Valdes et al.	Probe-based spectroscopy	6	LGG	-	82%	58%

^*From two different thresholds

For the neurosurgical oncologist, complete resection of lower-grade and non-enhancing tumors remains aparticular challenge. With the emergence of intraoperative florescence-guided resection using 5-ALA, the gap between LGG and HGG extent of resection may unfortunately continue to widen as WF microscopy has been to date robust primarily for visualizing higher-grade lesions. Initial experiences employing intraoperative spectroscopy and handheld microscope-based systems to visualize cellular 5-ALA tumor fluorescence suggests that infiltrating tumor cells can be identified within a LGG tumor mass and at the brain-tumor interface, including beyond the radiographically defined margins. Ongoing work is focused both on optimizing various high-resolution microscopic cellular detection/visualization strategies in the context of the neurosurgical workflow, as well as refining wide-field, quantitative imaging systems (e.g. spectroscopic) that are more sensitive for the detection of PpIX and therefore potentially of great utility to complement conventional low-resolution widefield assessments of PpIX fluorescence. Successful solutions will also need to be surgically intuitive, both in terms of physical deployment on patients and for data interpretation. In particular, these technologies will lead naturally to efforts in machine learning and computer vision to assist with image interpretation as well as integration with complementary operative tools such as MRI-based neuronavigation and intraoperative stimulation mapping.