Building a Two-Photon Microscope Is Easy

  • Spencer LaVere SmithEmail author
Part of the Neuromethods book series (NM, volume 148)


Building a two-photon microscope is easy because most of the work is done by the laser itself. All the microscope needs to do is to focus the laser light to a point, move it across the preparation, and measure the fluorescence photons emitted. These jobs are done by an objective, a scan engine, and a detector, respectively. That’s all there is to it.


Two-photon Multiphoton Optical design Laser scanning microscope Imaging Microscopy Design 


  1. 1.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805CrossRefGoogle Scholar
  2. 2.
    Heisenberg W (1927) Über den anschaulichen Inhalt der quantentheoretischen Kinematik und Mechanik. Zeitschrift für Physik 43(3–4):172–198CrossRefGoogle Scholar
  3. 3.
    Göppert-Mayer M (1931) Über Elementarakte mit zwei Quantensprüngen. Ann Phys (Leipzig) 9:273–294CrossRefGoogle Scholar
  4. 4.
    Yang MH, Abashin M, Saisan PA, Tian P, Ferri CG, Devor A, Fainman Y (2016) Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy. Opt Express 24(26):30173–30187. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76CrossRefGoogle Scholar
  6. 6.
    Masters BR, So PT (2004) Antecedents of two-photon excitation laser scanning microscopy. Microsc Res Tech 63(1):3–11. CrossRefPubMedGoogle Scholar
  7. 7.
    So PT, Dong CY, Masters BR, Berland KM (2000) Two-photon excitation fluorescence microscopy. Annu Rev Biomed Eng 2:399–429. CrossRefPubMedGoogle Scholar
  8. 8.
    Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18(3):351–357CrossRefGoogle Scholar
  9. 9.
    Moulton PF (1986) Spectroscopic and laser characteristics of Ti:Al2O3. OSAB: Opt Phys 3:125–133CrossRefGoogle Scholar
  10. 10.
    Podgorski K, Ranganathan G (2016) Brain heating induced by near-infrared lasers during multiphoton microscopy. J Neurophysiol 116(3):1012–1023. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kalies S, Kuetemeyer K, Heisterkamp A (2011) Mechanisms of high-order photobleaching and its relationship to intracellular ablation. Biomed Opt Express 2(4):805–816. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Macias-Romero C, Zubkovs V, Wang S, Roke S (2016) Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells. Biomed Opt Express 7(4):1458–1467. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ouzounov DG, Wang T, Wang M, Feng DD, Horton NG, Cruz-Hernandez JC, Cheng YT, Reimer J, Tolias AS, Nishimura N, Xu C (2017) In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain. Nat Methods 14(4):388–390. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fu W, Wright LG, Sidorenko P, Backus S, Wise FW (2018) Several new directions for ultrafast fiber lasers [Invited]. Opt Express 26(8):9432–9463. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Tsai PS, Kleinfeld D (2009) In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation. In: Frostig R (ed) Methods for in vivo optical imaging, vol 3. CRC Press, Boca Raton, pp 59–115Google Scholar
  16. 16.
    Sharafutdinova G, Holdsworth J, van Helden D (2010) Improved field scanner incorporating parabolic optics. Part 2: experimental verification and potential for volume scanning. Appl Opt 49(29):5517–5527. CrossRefPubMedGoogle Scholar
  17. 17.
    Sofroniew NJ, Flickinger D, King J, Svoboda K (2016) A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Elife 5.
  18. 18.
    Stirman JN, Smith IT, Kudenov MW, Smith SL (2016) Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain. Nat Biotechnol 34(8):857–862. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Tian X, Xu L, Li X, Shang G, Yao J (2010) Geometric distortion correction for sinusoidally scanned atomic force microscopic images. Paper presented at the IEEE International Conference on Imaging Systems and Techniques, Thessaloniki, 1–2 July 2010Google Scholar
  20. 20.
    Grewe BF, Voigt FF, van ‘t Hoff M, Helmchen F (2011) Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens. Biomed Opt Express 2(7):2035–2046. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gobel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4(1):73–79. CrossRefPubMedGoogle Scholar
  22. 22.
    Zurauskas M, Barnstedt O, Frade-Rodriguez M, Waddell S, Booth MJ (2017) Rapid adaptive remote focusing microscope for sensing of volumetric neural activity. Biomed Opt Express 8(10):4369–4379. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Shain WJ, Vickers NA, Goldberg BB, Bifano T, Mertz J (2017) Extended depth-of-field microscopy with a high-speed deformable mirror. Opt Lett 42(5):995–998. CrossRefPubMedGoogle Scholar
  24. 24.
    Kim KH, Buehler C, So PT (1999) High-speed, two-photon scanning microscope. Appl Opt 38(28):6004–6009CrossRefGoogle Scholar
  25. 25.
    Reddy GD, Kelleher K, Fink R, Saggau P (2008) Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat Neurosci 11(6):713–720. CrossRefPubMedCentralGoogle Scholar
  26. 26.
    Kirkby PA, Srinivas Nadella KM, Silver RA (2010) A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt Express 18(13):13721–13745. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Katona G, Szalay G, Maak P, Kaszas A, Veress M, Hillier D, Chiovini B, Vizi ES, Roska B, Rozsa B (2012) Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat Methods 9(2):201–208. CrossRefPubMedGoogle Scholar
  28. 28.
    Kong L, Tang J, Little JP, Yu Y, Lammermann T, Lin CP, Germain RN, Cui M (2015) Continuous volumetric imaging via an optical phase-locked ultrasound lens. Nat Methods 12(8):759–762. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Anselmi F, Ventalon C, Begue A, Ogden D, Emiliani V (2011) Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning. Proc Natl Acad Sci U S A 108(49):19504–19509. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Paluch-Siegler S, Mayblum T, Dana H, Brosh I, Gefen I, Shoham S (2015) All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation. Neurophotonics 2(3):031208. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Quirin S, Jackson J, Peterka DS, Yuste R (2014) Simultaneous imaging of neural activity in three dimensions. Front Neural Circuits 8:29. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Tsai PS, Mateo C, Field JJ, Schaffer CB, Anderson ME, Kleinfeld D (2015) Ultra-large field-of-view two-photon microscopy. Opt Express 23(11):13833–13847. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ji N, Freeman J, Smith SL (2016) Technologies for imaging neural activity in large volumes. Nat Neurosci 19(9):1154–1164. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Dunn AK, Wallace VP, Coleno M, Berns MW, Tromberg BJ (2000) Influence of optical properties on two-photon fluorescence imaging in turbid samples. Appl Opt 39(7):1194–1201CrossRefGoogle Scholar
  35. 35.
    Tung CK, Sun Y, Lo W, Lin SJ, Jee SH, Dong CY (2004) Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy. Microsc Res Tech 65(6):308–314. CrossRefPubMedGoogle Scholar
  36. 36.
    Schwertner M, Booth M, Wilson T (2004) Characterizing specimen induced aberrations for high NA adaptive optical microscopy. Opt Express 12(26):6540–6552CrossRefGoogle Scholar
  37. 37.
    Ohki K, Chung S, Kara P, Hubener M, Bonhoeffer T, Reid RC (2006) Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442(7105):925–928. CrossRefGoogle Scholar
  38. 38.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. CrossRefPubMedGoogle Scholar
  39. 39.
    Sheppard CJR, Castello M, Tortarolo G, Vicidomini G, Diaspro A (2017) Image formation in image scanning microscopy, including the case of two-photon excitation. J Opt Soc Am A Opt Image Sci Vis 34(8):1339–1350. CrossRefPubMedGoogle Scholar
  40. 40.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21(11):1369–1377. CrossRefPubMedGoogle Scholar
  41. 41.
    Lu R, Sun W, Liang Y, Kerlin A, Bierfeld J, Seelig JD, Wilson DE, Scholl B, Mohar B, Tanimoto M, Koyama M, Fitzpatrick D, Orger MB, Ji N (2017) Video-rate volumetric functional imaging of the brain at synaptic resolution. Nat Neurosci 20(4):620–628. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Theriault G, Cottet M, Castonguay A, McCarthy N, De Koninck Y (2014) Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging. Front Cell Neurosci 8:139. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Dufour P, Piche M, De Koninck Y, McCarthy N (2006) Two-photon excitation fluorescence microscopy with a high depth of field using an axicon. Appl Opt 45(36):9246–9252CrossRefGoogle Scholar
  44. 44.
    Prevedel R, Verhoef AJ, Pernia-Andrade AJ, Weisenburger S, Huang BS, Nobauer T, Fernandez A, Delcour JE, Golshani P, Baltuska A, Vaziri A (2016) Fast volumetric calcium imaging across multiple cortical layers using sculpted light. Nat Methods 13(12):1021–1028. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Scott BB, Brody CD, Tank DW (2013) Cellular resolution functional imaging in behaving rats using voluntary head restraint. Neuron 80(2):371–384. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Murphy TH, Boyd JD, Bolanos F, Vanni MP, Silasi G, Haupt D, LeDue JM (2016) High-throughput automated home-cage mesoscopic functional imaging of mouse cortex. Nat Commun 7:11611. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Visser TD, Oud JL (1994) Volume measurements in three-dimensional microscopy. Scanning 16:198–200CrossRefGoogle Scholar
  48. 48.
    Bor Z (1989) Distortion of femtosecond laser pulses in lenses. Opt Lett 14(2):119–121CrossRefGoogle Scholar
  49. 49.
    Busing L, Bonhoff T, Gottmann J, Loosen P (2013) Deformation of ultra-short laser pulses by optical systems for laser scanners. Opt Express 21(21):24475–24482. CrossRefPubMedGoogle Scholar
  50. 50.
    Oheim M, Beaurepaire E, Chaigneau E, Mertz J, Charpak S (2001) Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J Neurosci Meth 111(1):29–37Google Scholar
  51. 51.
    Singh A, McMullen JD, Doris EA, Zipfel WR (2015) Comparison of objective lenses for multiphoton microscopy in turbid samples. Biomed Opt Express 6(8):3113–3127. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Zinter JP, Levene MJ (2011) Maximizing fluorescence collection efficiency in multiphoton microscopy. Opt Express 19(16):15348–15362. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Engelbrecht CJ, Gobel W, Helmchen F (2009) Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection. Opt Express 17(8):6421–6435CrossRefGoogle Scholar
  54. 54.
    Ducros M, van 't Hoff M, Evrard A, Seebacher C, Schmidt EM, Charpak S, Oheim M (2011) Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing. J Neurosci Meth 198(2):172–180.
  55. 55.
    Michalet X, Cheng A, Antelman J, Suyama M, Arisaka K, Weiss S (2008) Hybrid photodetector for single-molecule spectroscopy and microscopy. Proc SPIE Int Soc Opt Eng 6862(68620F).
  56. 56.
    Fittinghoff D, Wiseman P, Squier J (2000) Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy. Opt Express 7(8):273–279CrossRefGoogle Scholar
  57. 57.
    Driscoll JD, Shih AY, Iyengar S, Field JJ, White GA, Squier JA, Cauwenberghs G, Kleinfeld D (2011) Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy. J Neurophysiol 105(6):3106–3113. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Amir W, Carriles R, Hoover EE, Planchon TA, Durfee CG, Squier JA (2007) Simultaneous imaging of multiple focal planes using a two-photon scanning microscope. Opt Lett 32(12):1731–1733CrossRefGoogle Scholar
  59. 59.
    Cheng A, Goncalves JT, Golshani P, Arisaka K, Portera-Cailliau C (2011) Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat Methods 8(2):139–142. CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Chen JL, Voigt FF, Javadzadeh M, Krueppel R, Helmchen F (2016) Long-range population dynamics of anatomically defined neocortical networks. Elife 5.
  61. 61.
    Lecoq J, Savall J, Vucinic D, Grewe BF, Kim H, Li JZ, Kitch LJ, Schnitzer MJ (2014) Visualizing mammalian brain area interactions by dual-axis two-photon calcium imaging. Nat Neurosci 17(12):1825–1829. CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Terada SI, Kobayashi K, Ohkura M, Nakai J, Matsuzaki M (2018) Super-wide-field two-photon imaging with a micro-optical device moving in post-objective space. Nat Commun 9(1):3550. CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Negrean A, Mansvelder HD (2014) Optimal lens design and use in laser-scanning microscopy. Biomed Opt Express 5(5):1588–1609. CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Young MD, Field JJ, Sheetz KE, Bartels RA, Squier J (2015) A pragmatic guide to multiphoton microscope design. Adv Opt Photonics 7(2):276–378. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.University of California Santa BarbaraSanta BarbaraUSA

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