Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection

Abstract

OBJECTIVE The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination. METHODS To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620-640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis. RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery. CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation. Clinical trial registration no.: NCT02191488 (clinicaltrials.gov).

Keywords: 5-aminolevulinic acid; ALA = 5-aminolevulinic acid; GBM = glioblastoma; PpIX = protoporphyrin IX; brain tumor; fluorescence-guided surgery; glioma; iSV = intraoperative stereovision; meningioma; metastasis; oncology; optical spectroscopy; protoporphyrin IX.

Figures

FIG. 1

Upper: The portion of the PpIX absorption (excitation) spectrum corresponding to red illumination is indicated by the shaded rectangle labeled “Excitation region.” The portion of the PpIX emission spectrum detected during red-light illumination (the longer wavelength region of 650–720 nm) is indicated by the hatched rectangle labeled “Detection region.” Lower: The absorption spectra of deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) showing the strong absorption in the blue wavelength region that limits tissue penetration. Figure is available in color online only.

FIG. 2

An adaptor (black arrow) attached at the objective lens of the operating microscope contains a 630 ± 10–nm bandpass filter (white arrow) that can be manually slid into and out of the xenon arc lamplight path of the microscope. Figure is available in color online only.

FIG. 3

Case 3. A: Axial T1-weighted MR image showing a right parietal tumor. B: Under white-light visualization, the intact and normal-appearing dura has been exposed upon elevation of the free bone flap. (The small yellow circle in the lower middle portion of the figure corresponds to the site of handheld optical probe measurements, whose spectra are shown in panel E.) C: Under blue-light excitation, no red fluorescence is visible. D: Under red-light excitation, fluorescence (650–720 nm) corresponding to underlying tumor was detected and is displayed in a false color overlaid on the white-light image. E: The optical spectra acquired with the handheld optical probe and corresponding model fits for PpIX, photoproducts, and autofluorescence at the site indicated by the small yellow circle in panel B. The typical red emission peak of PpIX at 635 nm under blue-light excitation is absent, while emission at longer wavelengths is detected under red-light excitation, confirming the presence of PpIX. F: Reconstruction of weighted MRI data corresponding to a plane 0–1 mm below the dural surface in the operative field. G: Reconstruction of weighted MRI data corresponding to a plane 0–5 mm below the dural surface in the operative field.

FIG. 4

Case 3. A: The cortical surface under white-light illumination. B: Under blue light, red fluorescence is evident in tumor-involved cortex. C: Under red light, longer-wavelength fluorescence represented in a false color is more extensive, corresponding to the contribution from subsurface PpIX. The location and distribution of fluorescence detected before dural opening (in Fig. 3) are consistent with those demonstrated with the dura open. D: Reconstruction of MRI data corresponding to a weighted average of the first 1 mm below the cortical surface. E: Reconstruction of weighted MRI data corresponding to the first 5 mm below the cortical surface.

FIG. 5

Case 11. A: Axial contrast-enhanced T1-weighted MR image of a well-demarcated tumor consistent with meningioma, arising from the floor of the right middle fossa. B: White-light image acquired upon initial exposure of the tumor (black arrow). C: Blue-light fluorescence image of the same surgical field. D: Overlay of fluorescence under red-light excitation. E: Reconstruction of MRI data corresponding to a weighted average of the first 1 mm below the surface of the surgical field. F: Reconstruction of weighted MRI data corresponding to the first 5 mm below the surface of the surgical field.

FIG. 6

Case 24. A: Axial 3D MPRAGE image obtained after gadolinium administration, showing a heterogeneously contrast-enhancing, left frontal mass lesion. B: White-light image of tumor-involved cortical surface. C: Blue-light excitation fluorescence image of the same surgical field demonstrating fluorescence in areas of obvious tumor involvement. D: Map of PpIX fluorescence detected during red-light illumination superposed on a white-light image of the surgical field. The small red circle in the upper left portion of the image corresponds to the region of interest interrogated with the handheld optical probe. E: Optical spectra and model fits for PpIX, photoproducts, and autofluorescence at the site indicated by the small red circle in panel D. The typical blue light–elicited PpIX emission peak at 635 nm is absent (confirming the absence of conventional red fluorescence signal under blue-light excitation), while emission at longer wavelengths detected under red-light excitation confirms the presence of PpIX.