Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in humans, accounting for 52% of all brain tumor cases.^{1, 2} The median survival is $12\text{to}15\text{months}$ . Optimal therapy consists of maximal safe surgical resection, followed by adjuvant chemoradiotherapy.³ Several studies demonstrate that the extent of surgical resection is a determinant of progression-free and overall survival.² Achieving a gross total resection is surgically challenging, because the normal and tumor-bearing brain can be similar in intraoperative appearance.³ A conservative resection by the surgeon can lead to suboptimal tumor debulking, whereas an aggressive technique may encroach on functionally significant brain. Various techniques have been employed to aid the neurosurgeon during tumor resection, including intraoperative MRI, neuronavigation, functional mapping via cortical stimulation, and ultrasonography.³ None of these techniques allows the direct pathologic discrimination of tissue.³

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.2 ns. We demonstrate a significant contrast in fluorescence lifetime between tumor $\left(1.77\pm 0.26\text{\hspace{0.17em} ns}\right)$ and normal $\left(2.50\pm 0.36\text{\hspace{0.17em} ns}\right)$ tissues at 450 nm and an over 80% intensity decrease at 390 nm emission in tumor versus normal areas. The time-resolved images were minimally affected by tissue morphology, endogenous absorbers, and illumination. These results demonstrate the potential of FLIM as an intraoperative diagnostic technique.

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (τ_HNSCC = 1.21 ± 0.04 ns) than the surrounding normal tissue (τ_N = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures.

We report a novel method for estimating fluorescence impulse response function (fIRF) from noise-corrupted time-domain fluorescence measurements of biological tissue. This method is based on the use of high-order Laguerre basis functions and a constrained least-squares approach that addresses the problem of overfitting due to increased model complexity. The new method was extensively evaluated on fluorescence data from simulation, fluorescent standard dyes, ex vivo tissue samples of atherosclerotic plaques and in vivo oral carcinoma. Current results demonstrate that this method allows for rapid and accurate deconvolution of multiple channel fluorescence decays without adaptively adjusting the Laguerre scale parameter. The appropriate choice of the scale parameter is essential for accurate estimation of the fIRF. The method described here is anticipated to play an important role in the development of computational techniques for real-time analysis of time-resolved fluorescence data from biological tissues and to support the advancement of fluorescence lifetime instrumentation for biomedical diagnostics by providing a means for on-line robust analysis of fluorescence decay.