Journal of Neuro-Oncology

, Volume 141, Issue 3, pp 495–505 | Cite as

Visualization technologies for 5-ALA-based fluorescence-guided surgeries

  • Linpeng WeiEmail author
  • David W. Roberts
  • Nader Sanai
  • Jonathan T. C. Liu
Topic Review



5-ALA-based fluorescence-guided surgery has been shown to be a safe and effective method to improve intraoperative visualization and resection of malignant gliomas. However, it remains ineffective in guiding the resection of lower-grade, non-enhancing, and deep-seated tumors, mainly because these tumors do not produce detectable fluorescence with conventional visualization technologies, namely, wide-field (WF) surgical microscopy.


We describe some of the main factors that limit the sensitivity and accuracy of conventional WF surgical microscopy, and then provide a survey of commercial and research prototypes being developed to address these challenges, along with their principles, advantages and disadvantages, as well as the current status of clinical translation for each technology. We also provide a neurosurgical perspective on how these visualization technologies might best be implemented for guiding glioma surgeries in the future.


Detection of PpIX expression in low-grade gliomas and at the infiltrative margins of all gliomas has been achieved with high-sensitivity probe-based visualization techniques. Deep-tissue PpIX imaging of up to 5 mm has also been achieved using red-light illumination techniques. Spectroscopic approaches have enabled more accurate quantification of PpIX expression.


Advancements in visualization technologies have extended the sensitivity and accuracy of conventional WF surgical microscopy. These technologies will continue to be refined to further improve the extent of resection in glioma patients using 5-ALA-induced fluorescence.


5-ALA PpIX Gliomas Intraoperative guidance Microscopy Spectroscopy 



We acknowledge funding support from the NIH, including grants from the NIDCR (R01 DE023497), the NCI (R01 CA175391), and the NINDS (R01 NS082745 and R01 NS052274).

Compliance with ethical standards

Conflict of interest

D.W.R. has equity in InSight Surgical Technologies LLC. L.W., N.S., and J.T.C.L. declare no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals.


  1. 1.
    Sanai N, Berger MS (2018) Surgical oncology for gliomas: the state of the art. Nat Rev Clin Oncol 15(2):112–125. CrossRefGoogle Scholar
  2. 2.
    Stummer W, van den Bent MJ, Westphal M (2011) Cytoreductive surgery of glioblastoma as the key to successful adjuvant therapies: new arguments in an old discussion. Acta Neurochir 153(6):1211–1218. CrossRefGoogle Scholar
  3. 3.
    Tonn JC, Stummer W (2008) Fluorescence-guided resection of malignant gliomas using 5-aminolevulinic acid: practical use, risks, and pitfalls. Clin Neurosurg 55:20–26Google Scholar
  4. 4.
    Price SJ, Gillard JH (2011) Imaging biomarkers of brain tumour margin and tumour invasion. Br J Radiol 84(Spec No 2):S159–S167. CrossRefGoogle Scholar
  5. 5.
    Aminolevulinic acid hydrochloride, known as ALA HCl (Gleolan, NX Development Corp.) as an optical imaging agent indicated in patients with gliomas (2017).
  6. 6.
    Gibbs SL, Chen B, O’Hara JA, Hoopes PJ, Hasan T, Pogue BW (2006) Protoporphyrin IX level correlates with number of mitochondria, but increase in production correlates with tumor cell size. Photochem Photobiol 82(5):1334–1341. CrossRefGoogle Scholar
  7. 7.
    Valdes PA, Kim A, Brantsch M, Niu C, Moses ZB, Tosteson TD, Wilson BC, Paulsen KD, Roberts DW, Harris BT (2011) delta-aminolevulinic acid-induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy. Neuro Oncol 13(8):846–856. CrossRefGoogle Scholar
  8. 8.
    Valdes PA, Leblond F, Kim A, Harris BT, Wilson BC, Fan XY, Tosteson TD, Hartov A, Ji SB, Erkmen K, Simmons NE, Paulsen KD, Roberts DW (2011) Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. J Neurosurg 115(1):11–17. CrossRefGoogle Scholar
  9. 9.
    Belykh E, Miller EJ, Hu D, Martirosyan NL, Woolf EC, Scheck AC, Byvaltsev VA, Nakaji P, Nelson LY, Seibel EJ, Preul MC (2018) Scanning fiber endoscope improves detection of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence at the boundary of infiltrative glioma. World Neurosurg 113:e51–e69. CrossRefGoogle Scholar
  10. 10.
    Stummer W, Tonn JC, Goetz C, Ullrich W, Stepp H, Bink A, Pietsch T, Pichlmeier U (2014) 5-Aminolevulinic acid-derived tumor fluorescence: the diagnostic accuracy of visible fluorescence qualities as corroborated by spectrometry and histology and postoperative imaging. Neurosurgery 74(3):310–319. discussion 319–320.CrossRefGoogle Scholar
  11. 11.
    Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ (2000) Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93(6):1003–1013. CrossRefGoogle Scholar
  12. 12.
    Panciani PP, Fontanella M, Schatlo B, Garbossa D, Agnoletti A, Ducati A, Lanotte M (2012) Fluorescence and image guided resection in high grade glioma. Clin Neurol Neurosurg 114(1):37–41. CrossRefGoogle Scholar
  13. 13.
    Yamada S, Muragaki Y, Maruyama T, Komori T, Okada Y (2015) Role of neurochemical navigation with 5-aminolevulinic acid during intraoperative MRI-guided resection of intracranial malignant gliomas. Clin Neurol Neurosurg 130:134–139. CrossRefGoogle Scholar
  14. 14.
    Roberts DW, Valdes PA, Harris BT, Fontaine KM, Hartov A, Fan X, Ji S, Lollis SS, Pogue BW, Leblond F, Tosteson TD, Wilson BC, Paulsen KD (2011) Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between delta-aminolevulinic acid-induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters. Clinical article. J Neurosurg 114(3):595–603. CrossRefGoogle Scholar
  15. 15.
    Diez Valle R, Tejada Solis S, Idoate Gastearena MA, Garcia de Eulate R, Dominguez Echavarri P, Aristu Mendiroz J (2011) Surgery guided by 5-aminolevulinic fluorescence in glioblastoma: volumetric analysis of extent of resection in single-center experience. J Neurooncol 102(1):105–113. CrossRefGoogle Scholar
  16. 16.
    Hefti M, von Campe G, Moschopulos M, Siegner A, Looser H, Landolt H (2008) 5-aminolevulinic acid induced protoporphyrin IX fluorescence in high-grade glioma surgery: a one-year experience at a single institutuion. Swiss Med Wkly 138(11–12):180–185. doi:2008/11/smw-12077Google Scholar
  17. 17.
    Acerbi F, Broggi M, Eoli M, Anghileri E, Cuppini L, Pollo B, Schiariti M, Visintini S, Orsi C, Franzini A, Broggi G, Ferroli P (2013) Fluorescein-guided surgery for grade IV gliomas with a dedicated filter on the surgical microscope: preliminary results in 12 cases. Acta Neurochir (Wien) 155(7):1277–1286. CrossRefGoogle Scholar
  18. 18.
    Tsugu A, Ishizaka H, Mizokami Y, Osada T, Baba T, Yoshiyama M, Nishiyama J, Matsumae M (2011) Impact of the combination of 5-aminolevulinic acid-induced fluorescence with intraoperative magnetic resonance imaging-guided surgery for glioma. World Neurosurg 76(1–2):120–127. CrossRefGoogle Scholar
  19. 19.
    Hadjipanayis CG, Widhalm G, Stummer W (2015) What is the Surgical Benefit of Utilizing 5-Aminolevulinic Acid for Fluorescence-Guided Surgery of Malignant Gliomas? Neurosurgery 77(5):663–673. CrossRefGoogle Scholar
  20. 20.
    Rapp M, Kamp M, Steiger HJ, Sabel M (2014) Endoscopic-assisted visualization of 5-aminolevulinic acid-induced fluorescence in malignant glioma surgery: a technical note. World Neurosurg 82(1–2):E277–E279. CrossRefGoogle Scholar
  21. 21.
    Belloch JP, Rovira V, Llacer JL, Riesgo PA, Cremades A (2014) Fluorescence-guided surgery in high grade gliomas using an exoscope system. Acta Neurochir (Wien) 156(4):653–660. CrossRefGoogle Scholar
  22. 22.
    Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ, Group AL-GS (2006) Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol 7(5):392–401. CrossRefGoogle Scholar
  23. 23.
    Sanai N, Snyder LA, Honea NJ, Coons SW, Eschbacher JM, Smith KA, Spetzler RF (2011) Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas. J Neurosurg 115(4):740–748. CrossRefGoogle Scholar
  24. 24.
    Valdes PA, Jacobs V, Harris BT, Wilson BC, Leblond F, Paulsen KD, Roberts DW (2015) Quantitative fluorescence using 5-aminolevulinic acid-induced protoporphyrin IX biomarker as a surgical adjunct in low-grade glioma surgery. J Neurosurg 123(3):771–780. CrossRefGoogle Scholar
  25. 25.
    Valdes PA, Leblond F, Jacobs VL, Wilson BC, Paulsen KD, Roberts DW (2012) Quantitative, spectrally-resolved intraoperative fluorescence imaging. Sci Rep 2:798. CrossRefGoogle Scholar
  26. 26.
    Richter JCO, Haj-Hosseini N, Hallbeck M, Wardell K (2017) Combination of hand-held probe and microscopy for fluorescence guided surgery in the brain tumor marginal zone. Photodiagnosis Photodyn Ther 18:185–192. CrossRefGoogle Scholar
  27. 27.
    Konecky SD, Owen CM, Rice T, Valdes PA, Kolste K, Wilson BC, Leblond F, Roberts DW, Paulsen KD, Tromberg BJ (2012) Spatial frequency domain tomography of protoporphyrin IX fluorescence in preclinical glioma models. J Biomed Opt 17(5):056008. CrossRefGoogle Scholar
  28. 28.
    Kepshire DS, Gibbs-Strauss SL, O’Hara JA, Hutchins M, Mincu N, Leblond F, Khayat M, Dehghani H, Srinivasan S, Pogue BW (2009) Imaging of glioma tumor with endogenous fluorescence tomography. J Biomed Opt 14(3):030501. CrossRefGoogle Scholar
  29. 29.
    Kim A, Roy M, Dadani FN, Wilson BC (2010) Topographic mapping of subsurface fluorescent structures in tissue using multiwavelength excitation. J Biomed Opt 15(6):066026. CrossRefGoogle Scholar
  30. 30.
    Leblond F, Ovanesyan Z, Davis SC, Valdes PA, Kim A, Hartov A, Wilson BC, Pogue BW, Paulsen KD, Roberts DW (2011) Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging. Phys Med Biol 56(21):6823–6837. CrossRefGoogle Scholar
  31. 31.
    Kolste KK, Kanick SC, Valdes PA, Jermyn M, Wilson BC, Roberts DW, Paulsen KD, Leblond F (2015) Macroscopic optical imaging technique for wide-field estimation of fluorescence depth in optically turbid media for application in brain tumor surgical guidance. J Biomed Opt 20(2):26002. CrossRefGoogle Scholar
  32. 32.
    Jermyn M, Kolste K, Pichette J, Sheehy G, Angulo-Rodriguez L, Paulsen KD, Roberts DW, Wilson BC, Petrecca K, Leblond F (2015) Macroscopic-imaging technique for subsurface quantification of near-infrared markers during surgery. J Biomed Opt 20(3):036014. CrossRefGoogle Scholar
  33. 33.
    Roberts DW, Olson JD, Evans LT, Kolste KK, Kanick SC, Fan X, Bravo JJ, Wilson BC, Leblond F, Marois M, Paulsen KD (2018) Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection. J Neurosurg 128(6):1690–1697. CrossRefGoogle Scholar
  34. 34.
    Meza D, Wang D, Wang Y, Borwege S, Sanai N, Liu JT (2015) Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections. Lasers Surg Med 47(4):289–295. CrossRefGoogle Scholar
  35. 35.
    Stummer W, Stocker S, Wagner S, Stepp H, Fritsch C, Goetz C, Goetz AE, Kiefmann R, Reulen HJ (1998) Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery 42(3):518–525 (discussion 525–516)CrossRefGoogle Scholar
  36. 36.
    Stummer W, Stepp H, Moller G, Ehrhardt A, Leonhard M, Reulen HJ (1998) Technical principles for protoporphyrin-IX-fluorescence guided microsurgical resection of malignant glioma tissue. Acta Neurochir (Wien) 140(10):995–1000CrossRefGoogle Scholar
  37. 37.
    Liu JT, Meza D, Sanai N (2014) Trends in fluorescence image-guided surgery for gliomas. Neurosurgery 75(1):61–71. CrossRefGoogle Scholar
  38. 38.
    Valdes PA, Roberts DW, Lu FK, Golby A (2016) Optical technologies for intraoperative neurosurgical guidance. Neurosurg Focus 40(3):E8. CrossRefGoogle Scholar
  39. 39.
    Pogue BW, Gibbs-Strauss S, Valdes PA, Samkoe K, Roberts DW, Paulsen KD (2010) Review of Neurosurgical Fluorescence Imaging Methodologies. IEEE J Sel Top Quantum Electron 16(3):493–505. CrossRefGoogle Scholar
  40. 40.
    Belykh E, Martirosyan NL, Yagmurlu K, Miller EJ, Eschbacher JM, Izadyyazdanabadi M, Bardonova LA, Byvaltsev VA, Nakaji P, Preul MC (2016) Intraoperative fluorescence imaging for personalized brain tumor resection: current state and future directions. Front Surg 3:55. CrossRefGoogle Scholar
  41. 41.
    Tamura Y, Kuroiwa T, Kajimoto Y, Miki Y, Miyatake S, Tsuji M (2007) Endoscopic identification and biopsy sampling of an intraventricular malignant glioma using a 5-aminolevulinic acid-induced protoporphyrin IX fluorescence imaging system. Technical note. J Neurosurg 106(3):507–510. CrossRefGoogle Scholar
  42. 42.
    Haj-Hosseini N, Richter J, Andersson-Engels S, Wårdell K (2009) Photobleaching behavior of protoporphyrin IX during 5-aminolevulinic acid marked glioblastoma detection. Proc. SPIE 7161, Photonic Therapeutics and Diagnostics V, 716131.
  43. 43.
    Potapov AA, Usachev DJ, Loshakov VA, Cherekaev VA, Kornienko VN, Pronin IN, Kobiakov GL, Kalinin PL, Gavrilov AG, Stummer W, Golbin DA, Zelenkov PV (2008) First experience in 5-ALA fluorescence-guided and endoscopically assisted microsurgery of brain tumors. Med Laser Appl 23(4):202–208. CrossRefGoogle Scholar
  44. 44.
    Haj-Hosseini N, Richter J, Andersson-Engels S, Wardell K (2010) Optical touch pointer for fluorescence guided glioblastoma resection using 5-aminolevulinic acid. Lasers Surg Med 42(1):9–14. CrossRefGoogle Scholar
  45. 45.
    Kim A, Khurana M, Moriyama Y, Wilson BC (2010) Quantification of in vivo fluorescence decoupled from the effects of tissue optical properties using fiber-optic spectroscopy measurements. J Biomed Opt 15(6):067006CrossRefGoogle Scholar
  46. 46.
    Ishihara R, Katayama Y, Watanabe T, Yoshino A, Fukushima T, Sakatani K (2007) Quantitative spectroscopic analysis of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence intensity in diffusely infiltrating astrocytomas. Neurol Med Chir (Tokyo) 47(2):53–57; discussion 57CrossRefGoogle Scholar
  47. 47.
    Utsuki S, Oka H, Sato S, Suzuki S, Shimizu S, Tanaka S, Fujii K (2006) Possibility of using laser spectroscopy for the intraoperative detection of nonfluorescing brain tumors and the boundaries of brain tumor infiltrates—Technical note. J Neurosurg 104(4):618–620. doi: CrossRefGoogle Scholar
  48. 48.
    Utzinger U, Richards-Kortum RR (2003) Fiber optic probes for biomedical optical spectroscopy. J Biomed Opt 8(1):121–147. CrossRefGoogle Scholar
  49. 49.
    Valdes PA, Jacobs VL, Wilson BC, Leblond F, Roberts DW, Paulsen KD (2013) System and methods for wide-field quantitative fluorescence imaging during neurosurgery. Opt Lett 38(15):2786–2788. CrossRefGoogle Scholar
  50. 50.
    Bravo JJ, Olson JD, Davis SC, Roberts DW, Paulsen KD, Kanick SC (2017) Hyperspectral data processing improves PpIX contrast during fluorescence guided surgery of human brain tumors. Sci Rep 7(1):9455. CrossRefGoogle Scholar
  51. 51.
    Markwardt NA, Haj-Hosseini N, Hollnburger B, Stepp H, Zelenkov P, Ruhm A (2016) 405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence-guided stereotactic biopsy of brain tumors. J Biophotonics 9(9):901–912. CrossRefGoogle Scholar
  52. 52.
    Cuccia DJ, Bevilacqua F, Durkin AJ, Tromberg BJ (2005) Modulated imaging: quantitative analysis and tomography of turbid media in the spatial-frequency domain. Opt Lett 30(11):1354–1356CrossRefGoogle Scholar
  53. 53.
    Wirth D, Kolste K, Kanick S, Roberts DW, Leblond F, Paulsen KD (2017) Fluorescence depth estimation from wide-field optical imaging data for guiding brain tumor resection: a multi-inclusion phantom study. Biomed Opt Express 8(8):3656–3670. CrossRefGoogle Scholar
  54. 54.
    Wei L, Chen Y, Yin C, Borwege S, Sanai N, Liu JTC (2017) Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology. J Biomed Opt 22(4):46005. CrossRefGoogle Scholar
  55. 55.
    Yin C, Glaser AK, Leigh SY, Chen Y, Wei L, Pillai PC, Rosenberg MC, Abeytunge S, Peterson G, Glazowski C, Sanai N, Mandella MJ, Rajadhyaksha M, Liu JT (2016) Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology. Biomed Opt Express 7(2):251–263. CrossRefGoogle Scholar
  56. 56.
    Wei L, Yin C, Liu JTC (2019) Dual-axis confocal microscopy for point-of-care pathology. IEEE J Sel Top Quantum Electron 25(1):1–10. Google Scholar
  57. 57.
    Liu JT, Loewke NO, Mandella MJ, Levenson RM, Crawford JM, Contag CH (2011) Point-of-care pathology with miniature microscopes. Anal Cell Pathol (Amst) 34(3):81–98. CrossRefGoogle Scholar
  58. 58.
    Gmitro AF, Aziz D (1993) Confocal microscopy through a fiber-optic imaging bundle. Opt Lett 18(8):565–567. CrossRefGoogle Scholar
  59. 59.
    Descour RR-KLSSBR (2002) Fiber-optic confocal imaging apparatus and methods of use. US6370422B1.
  60. 60.
    Udovich JA, Kirkpatrick ND, Kano A, Tanbakuchi A, Utzinger U, Gmitro AF (2008) Spectral background and transmission characteristics of fiber optic imaging bundles. Appl Opt 47(25):4560–4568. CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Mechanical EngineeringUniversity of WashingtonSeattleUSA
  2. 2.Section of NeurosurgeryDartmouth-Hitchcock Medical CenterLebanonUSA
  3. 3.Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical CenterLebanonUSA
  4. 4.Thayer School of EngineeringDartmouth CollegeHanoverUSA
  5. 5.Geisel School of MedicineDartmouth CollegeHanoverUSA
  6. 6.Department of Neurological SurgeryBarrow Neurological InstitutePhoenixUSA
  7. 7.Department of PathologyUniversity of WashingtonSeattleUSA

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