First in patient assessment of brain tumor infiltrative margins using simultaneous time-resolved measurements of 5-ALA-induced PpIX fluorescence and tissue autofluorescence

Alba Alfonso-García, Xiangnan Zhou, Julien Bec, Silvia N. Anbunesan, Farzad Fereidouni, Lee Way Jin, Han S. Lee, Orin Bloch, Laura Marcu

Research output: Contribution to journalArticlepeer-review


SIGNIFICANCE: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection. AIM: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients. APPROACH: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33  ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation. RESULTS: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τPpIX       ∼  3 to 14 ns, τNAD(P)H  =  3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration. CONCLUSIONS: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Original languageEnglish (US)
JournalJournal of Biomedical Optics
Issue number2
StatePublished - Feb 1 2022
Externally publishedYes


  • 5-ALA-induced PpIX fluorescence
  • brain tumor
  • fluorescence lifetime imaging
  • glioblastoma
  • nicotinamide adenine (phosphate) dinucleotide

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Biomaterials
  • Biomedical Engineering


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