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Electron Microscopic Measurement of the Size of the Optical Focus in Laser Scanning Microscopy

Published online by Cambridge University Press:  08 May 2012

Alison McDonald ,
William B. Amos  and
Gail McConnell
Alison McDonald*
Affiliation:
Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
William B. Amos
Affiliation:
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
Gail McConnell
Affiliation:
Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
*
Corresponding author. E-mail: alison.mcdonald@strath.ac.uk
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Abstract

We describe a method for measuring the lateral focal spot size of a multiphoton laser scanning microscope (LSM) with unprecedented accuracy. A specimen consisting of an aluminum film deposited on a glass coverslip was brought into focus in a LSM and the laser intensity was then increased enough to perform nanoablation of the metal film. This process leaves a permanent trace of the raster path usually taken by the beam during the acquisition of an optical image. A scanning electron microscope (SEM) was then used to determine the nanoablated line width to high accuracy, from which the lateral spot size and hence resolution of the LSM can be determined. To demonstrate our method, we performed analysis of a multiphoton LSM at various infrared wavelengths, and we report measurements of optical lateral spot size with an accuracy of 20 nm, limited only by the resolution of the SEM.

Keywords

resolutionlaser scanning microscopySEMablation
Type
Techniques Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 3 , June 2012 , pp. 596 - 602
Copyright
Copyright © Microscopy Society of America 2012

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References

Abbe, E. (1884). A note on the proper definition of the amplifying power of a lens system. J Royal Microsc Soc 4, 348–351.CrossRefGoogle Scholar
Amos, W. & White, J. (1987). Use of confocal imaging in the study of biological structures. Appl Opt 26, 3239–3243.CrossRefGoogle Scholar
Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwarz, J. & Hess, H.F. (2006). Imaging intracellular fluorescent proteins at nanometre resolution. Science 313, 1642–1645.CrossRefGoogle Scholar
Cox, G. & Sheppard, C.J.R. (2004). Practical limits of resolution in confocal and non-linear microscopy. Microsc Res Tech 63, 18–22.CrossRefGoogle ScholarPubMed
Denk, W., Strickler, J.H. & Webb, W.W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248, 73.CrossRefGoogle ScholarPubMed
Ditchburn, R.W. (1991). Light. New York: Dover Publications Inc.Google Scholar
Gordon, M.P., Ha, T. & Selvin, P.R. (2004). Single-molecule high-resolution imaging with photobleaching. PNAS 101, 6462–6465.CrossRefGoogle ScholarPubMed
Hell, S.W. & Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Opt Lett 19, 780–782.CrossRefGoogle ScholarPubMed
Inoué, S. (2006). Foundations of confocal scanned imaging in light microscopy. In Handbook of Biological Confocal Microscopy, Pawley, J. (Ed.), pp. 1–19. New York: Springer.Google Scholar
Jee, Y., Becker, M.F. & Walser, R.M. (1988). Laser-induced damage on single-crystal metal surfaces. J Opt Soc Am B 5, 648–659.CrossRefGoogle Scholar
Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. (2000). Fluorescence microscopy with diffraction barrier broken by stimulated emission. PNAS 18, 8206–8210.CrossRefGoogle Scholar
Liang, F., Vallee, R., Gingras, D. & Chin, S.L. (2011). Role of ablation and incubation processes on surface nanograting formation. Opt Mat Exp 1, 1244–1250.CrossRefGoogle Scholar
Liu, J.M. (1982). Simple technique for measurements of pulsed Gaussian-beam spot sizes. Opt Lett 7, 196–198.CrossRefGoogle ScholarPubMed
Liu, X., Du, D. & Mourou, G. (1997). Laser ablation and micromachining with ultrashort laser pulses. IEEE J Quantum Elec 33, 1706–1716.CrossRefGoogle Scholar
Mittman, W., Wallace, D.J., Czubayko, U., Herb, J.T., Schaefer, A.T., Looger, L.L., Denk, W. & Kerr, J.N.D. (2011). Two-photon calcium imaging of evoked activity from L5 somatosenory neurons in vivo. Nat Neurosci 14, 1089–1093.CrossRefGoogle Scholar
Muller, M., Schmidt, J.R., Mironov, S.L. & Richter, D.W. (2003). Construction and performance of a custom-built two-photon laser scanning system. App Phys D 36, 1747–1757.CrossRefGoogle Scholar
Oldenbourg, R., Terada, H., Tiberio, R. & Inoué, S. (1993). Image sharpness and contrast transfer in coherent confocal microscopy. J Microsc 172, 31–39.CrossRefGoogle ScholarPubMed
Sheppard, C. & Kompfner, R. (1978). Resonant scanning optical microscope. App Opt 17, 2879–2882.CrossRefGoogle ScholarPubMed
Siegman, A.E., Nemes, G. & Serna, J. (1998). How to (maybe) measure laser beam quality. In DPSS (Diode Pumped Solid State Laser): Applications and Issues, Dowley, M. (Ed.), p. MQ1. Washington, DC: Optical Society of America.Google Scholar
Tan, B., Dalili, A. & Venkatakrishnan, K. (2008). High repetition rate femtosecond laser nano-machining of thin films. Appl Phys A 95, 537–545.CrossRefGoogle Scholar
Turner, G.L.E & Bradbury, S. (1966). An electron microscopical examination of Norbert's finest test-plate of twenty bands. J Royal Microsc Soc 85, 435–447.CrossRefGoogle Scholar
Wang, C., Qiao, L., He, F., Cheng, Y. & Xu, Z. (2011). Extension of imaging depth in two-photon fluorescence microscopy using a long-wavelength high-pulse-energy femtosecond laser source. J Microsc 243, 179–183.CrossRefGoogle ScholarPubMed
Zucker, R.M. (2006). Evaluation of confocal microscopy system performance. Methods Mol Biol 319, 77–135.CrossRefGoogle ScholarPubMed