Quantitative, spectrally-resolved intraoperative fluorescence imaging - PubMed (original) (raw)
Quantitative, spectrally-resolved intraoperative fluorescence imaging
Pablo A Valdés et al. Sci Rep. 2012.
Abstract
Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field.
Conflict of interest statement
The authors declare a US provisional patent application serial No. 61/588,708 on some aspects of the technology used in this study.
Figures
Figure 1. Wide-field image estimate versus true PpIX concentration, C_PpIX_, in phantoms with different absorption and scattering optical properties (see Table 1).
(A) Pixel-averaged qFI estimates of C_PpIX_. (B) Coefficient of variation, COV, across the FOV. (C) Area under the raw optical signal (fluorescence intensity) curve from 610–720 nm. R2 = coefficient of determination; mPE = mean percentage error; mCOV = mean coefficient of variation.
Figure 2. Wide-field image estimate versus changing PpIX concentration, CPpIX, in phantoms with different fluorescein concentrations (10, 5, 2.5, 1.25 μg/ml).
(A) Pixel-averaged qFI estimates of C_PpIX_. (B) Coefficient of variation, COV, across the FOV. Multiple points per CPpIX represent different fluorescein concentrations.
Figure 3. Wide-field qFI estimates of CPpIX in a rodent model of glioma.
(A) White light FOV. (B) Corresponding vFI demonstrating red PpIX fluorescence in the tumor bulk. (C) Map of qFI estimates of C_PpIX_. (D) Corresponding image overlay demonstrating C_PpIX_ >150 ng/ml in the tumor bulk, as well as significant (C_PpIX_ ~100 ng/ml) PpIX in the infiltrating margin (white arrow). Color bar values are in ng/ml.
Figure 4. Wide-field qFI in the presence of two fluorophores in a rodent model of glioma.
(A) White light FOV. (B) Corresponding vFI demonstrates green fluorescein fluorescence in the tumor bulk, and no visible PpIX red fluorescence. (C) Map of qFI estimates of C_PpIX_. (D) Corresponding image overlay shows C_PpIX_ >200 ng/ml in the tumor bulk. (E) Map of normalized fluorescein levels. (F) Corresponding image overlay demonstrates fluorescein levels in the tumor bulk as well as diffusely scattered through normal brain. Significant PpIX concentration (C_PpIX_ ~100 ng/ml) was found in the infiltrating margin (white arrow in C) with minimal fluorescein present. Spectral decomposition (far right) indicates the varying contributions (at arrow locations in B marked +, top spectral plot, and *, bottom spectral plot) from each fluorophore in the FOV. Color bar values for C and D are in ng/ml and for E and F in normalized arbitrary units.
Figure 5. Wide-field qFI during a human GBM surgery.
Images at the beginning (A–C), near the end (E–G), and at the end of the case (I–K) show vFI, white light, and white light+qFI views, respectively. High levels of PpIX are evident at the start of surgery (in B), whereas near the end of surgery, no PpIX is noted on vFI (in F), but significant PpIX was found in qFI near the center of the FOV (in G) with histological corroboration as tumor (in L). At the end of surgery, no significant PpIX levels remained in the qFI acquisition (in K). Spectra obtained at the beginning of surgery (in D) from three locations (1 = vFI positive tumor, red mark in A; 2 = qFI positive/vFI negative tumor, blue mark in A; 3 = normal brain, green mark in A), and spectrum acquired near the end of surgery (in H) from one location (1 = qFI positive/vFI negative tumor, blue mark in E) are shown.
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