Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery
Abstract
In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-ofmagnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival.
1. Introduction
The goal of neurosurgery in brain tumor management is to maximize the extent of tumor resection while minimizing functional impairment secondary to surgery. Standard surgical equipment comprises a white-light neurosurgical microscope and a neuronavigation unit to guide surgery based on preoperative magnetic resonance imaging (MRI). The use of preoperative MRI improves the accuracy and safety of surgical resections using spatial coregistration of MRI scans with intraoperative magnified bright-field images taken in real time during surgery. Indeed, image-guided neurosurgery has increased the completeness of tumor resection as determined by comparing tumor tissue visible in preoperative relative to postoperative MRI [1].
More recently, intraoperative fluorescence imaging based on the optical contrast of molecules such as indocyanine green, fluorescein and protoporphyrin IX (PpIX) has emerged [2]. PpIX is synthesized and retained preferentially in tumor cells following administration of the pro-drug 5-aminolevulinic acid (ALA). The biological origin of the optical contrast varies between fluorophores, each providing different and potentially complementary clinical information. For example, preferential accumulation of PpIX in tumor cells results in part from differences in heme biosynthesis and pro-drug uptake. Glioblastoma resection surgery guided by the red fluorescence of PpIX upon violet light excitation has been demonstrated in controlled clinical trials to improve the completeness of tumor resection relative to resections based solely on bright-field, white-light visualization [3].
For the majority of clinical studies of ALA-PpIX guided resection of brain tumors, fluorescence detection is achieved with a commercial neurosurgical microscope [4]. The emitted fluorescence is detected by a color RGB camera or by the naked eye through the oculars of the microscope. The resection is thus guided by subjective visual assessment of the fluorescence images in real time, referred to as qualitative fluorescence imaging.
Clinical reports have suggested that the metabolic marker PpIX aids in surgical guidance of other brain pathologies, including meningioma and metastatic brain lesions [5–8]. It has also been used in other organs for tumor localization and treatment guidance [9]. However, qualitative fluorescence imaging is intrinsically limited in sensitivity and specificity. The sensitivity, i.e. the ability to detect (residual) tumor, is hampered by the limited detection efficiency of CCD cameras (e.g., read-out noise, quantum efficiency), the presence of the leakage reflectance signals into the detection path (background), and the confounding effects of intrinsic tissue optical properties [6]. Specificity is negatively impacted by the lack of spectral resolution when using high-pass interference filtering. Importantly, this limitation prevents subtraction of tissue auto-fluorescence background, which places a lower limit on the concentration of the exogenous fluorophore that can be detected and does not allow simultaneous imaging of multiple fluorophores. Moreover, studies using point fluorescence measurements combined with diffuse reflectance spectroscopy suggest that low-grade gliomas also produce diagnostic levels of ALA-induced PpIX but that the signals are too low to be detected with standard qualitative wide-field fluorescence imaging [6,10]. Patients with glioblastomas (WHO grade 4 gliomas [11]) do see some survival benefits based on maximizing extent of resection [12,13] and complete resection is a major factor in preventing recurrence and improving survival for grade 4 gliomas [14].
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