Flavin fluorescence lifetime and autofluorescence optical redox ratio for improved visualization and classification of brain tumors.

Purpose: Modern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated. Experimental design: Fluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3). Results: Protein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification. Conclusions: Our results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery.

Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

Corrigendum: Quantitative Modulation of PpIX Fluorescence and Improved Glioma Visualization.

[This corrects the article DOI: 10.3389/fsurg.2019.00041.].

Towards real-time wide-field fluorescence lifetime imaging of 5-ALA labeled brain tumors with multi-tap CMOS cameras.

Fluorescence guided neurosurgery based on 5-aminolevulinic acid (5-ALA) has significantly increased maximal safe resections. Fluorescence lifetime imaging (FLIM) of 5-ALA could further boost this development by its increased sensitivity. However, neurosurgeons require real-time visual feedback which was so far limited in dual-tap CMOS camera based FLIM. By optimizing the number of phase frames required for reconstruction, we here demonstrate real-time 5-ALA FLIM of human high- and low-grade glioma with up to 12 Hz imaging rate over a wide field of view (11.0 x 11.0 mm). Compared to conventional fluorescence imaging, real-time FLIM offers enhanced contrast of weakly fluorescent tissue.

Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery.

SIGNIFICANCE: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed. AIM: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging. APPROACH: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology. RESULTS: Our imaging system enabled macroscopic FLIM of a 6.5 × 6.5 mm2 field of view at spatial resolutions <20 µm. A frame of 512 × 512 pixels with a lifetime accuracy <1 ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology. CONCLUSIONS: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

Quantitative Modulation of PpIX Fluorescence and Improved Glioma Visualization.

5-Aminolevulinic acid (5-ALA) induced fluorescence to augment surgical resection for high grade glioma has become a standard of care. Protoporphyrin IX (PpIX) visibility is however subject to the variability of the single tumor expression and to the interobserver interpretation. We therefore hypothesized that in different glioma cell lines with variable 5-ALA induced fluorescence, the signal can be pharmacologically increased. We therefore analyzed in three different GBM cell lines, with different expression of epidermal growth factor receptor (EGFR), the variability of 5-ALA induced PpIX fluorescence after the pharmacological blockade at different steps of PpIX breakdown and influencing the outbound transport of PpIX. Using flow cytometry, fluorescence microplate reader, and confocal microscopy the PpIX fluorescence was analyzed after exposure to tin protoporphyrin IX (SnPP), deferoxamine (DFO), and genistein. We furthermore constructed a microscope (Qp9-microscope) being able to measure quantitatively the concentration of PpIX. These values were compared with the extraction of PpIX in tumor biopsy taken during the GBM surgery. Although all three cell lines showed an increase to 5-ALA induced fluorescence their baseline activity was different. Treatment with either SnPP, DFO and genistein was able to increase 5-ALA induced fluorescence. Qp9-microscopy of tumor sample produced a color coded PpIX concentration map which was overlaid on the tumor image. The PpIX extraction from tumor sample analyzed using the plate reader gave lower values of the concentration, as compared to the expected values of the Qp9-microscope, however still in the same decimal range of µg/mL. This may be due to homogenization of the values during extraction and cell disaggregation. In conclusion pharmacological augmentation in GBM cell lines of PpIX signal is possible. A quantitative PpIX map for surgery is feasible and may help refine surgical excision. Further correlations of tumor tissue samples and Qp9-microscopy is needed, prior to develop an intraoperative surgical adjunct to the already existing 5-ALA induced surgery.

Simple integrated single-atom detector.

We present a reliable and robust integrated fluorescence detector capable of detecting single atoms. The detector consists of a tapered lensed single-mode fiber for precise delivery of excitation light and a multimode fiber to collect the fluorescence. Both are mounted in lithographically defined SU-8 holding structures on an atom chip. 87Rb atoms propagating freely in a magnetic guide are detected with an efficiency of up to 66%, and a signal-to-noise ratio in excess of 100 is obtained for short integration times.

Fabrication of alignment structures for a fiber resonator by use of deep-ultraviolet lithography.

We present a novel method to mount and align an optical-fiber-based resonator on the flat surface of an atom chip with ultrahigh precision. The structures for mounting a pair of fibers, which constitute the fiber resonator, are produced by a spin-coated SU-8 photoresist technique by use of deep-UV lithography. The design and production of the SU-8 structures are discussed. From the measured finesses we calculate the coupling loss of the SU-8 structures acting as a kind of fiber splice to be smaller than 0.013 dB.