Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence‐guided neurosurgery: An ex vivo feasibility study

Abstract

Achieving a maximal safe extent of resection during brain tumor surgery is the goal for improved patient prognosis. Fluorescence‐guided neurosurgery using 5‐aminolevulinic acid (5‐ALA) induced protoporphyrin IX has thereby become a valuable tool enabling a high frequency of complete resections and a prolonged progression‐free survival in glioblastoma patients. We present a widefield fluorescence lifetime imaging device with 250 mm working distance, working under similar conditions such as surgical microscopes based on a time‐of‐flight dual tap CMOS camera. In contrast to intensity‐based fluorescence imaging, our method is invariant to light scattering and absorption while being sensitive to the molecular composition of the tissue. We evaluate the feasibility of lifetime imaging of protoporphyrin IX using our system to analyze brain tumor phantoms and fresh 5‐ALA‐labeled human tissue samples. The results demonstrate the potential of our lifetime sensing device to go beyond the limitation of current intensity‐based fluorescence‐guided neurosurgery.

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

“…For PpIX, a custom-made reference sample embedded in polymethylmethacrylate (Starna Scientific Ltd., Ilford, United Kingdom) was measured. 8 The measured lifetimes agreed within the range of error margins with reported literature values for the dyes in the respective solvent ( Table 1). The strongest measured lifetime deviation respective to literature (0.2 ns) was measured for fluorescein, which could be caused by the pH dependence of fluorescein lifetime.…”

“…A detailed study design can be found in our previous publication. 8 Tissue samples were imaged within an hour after resection and kept moist using artificial cerebrospinal fluid. For the first patient, the surgeon did not report any visible PpIX fluorescence.…”

“…Both specimen imaged with our system showed areas with lifetimes <2 ns, which corresponded well to the range of reported autofluorescence lifetimes of tumor-adjacent physiological brain parenchyma. 11 Increased lifetimes clearly indicated the accumulation of PpIX, 8,11 which enabled more sensitive tumor delineation than fluorescence intensity. This was supported by the fact that both tissue samples either had blood vessels or hemorrhages.…”

What scans we will read: imaging instrumentation trends in clinical oncology

“…Metabolites, such as Flavin adenine dinucleotide (FAD) or Nicotinamide adenine dinucleotide (NADH and the redox partner NAD+) have been proven valuable endogenous molecular probes of cell metabolism. Recent developments in fluorescence lifetime imaging (FLIM) show significant changes of FAD and NADH lifetime in tumour versus healthy tissue [207,208]. Fluorophores are excited in the short visible wavelength range where scattering limits large penetration depths.…”

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

“…Recently, our group developed a frequency domain FLIM system [21] around a dual-tap complementary metal-oxide semiconductor (CMOS) camera [22,23] with similar working distance and field of view (FOV) as found in modern surgical microscopes. To reconstruct the fluorescence lifetime, the camera generates several images with a phase-shifted excitation for each frame to cover a full circle of 360 degrees.…”