Microscopy and Microanalysis

Techniques Development

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

Alison McDonalda1 c1, William B. Amosa2 and Gail McConnella1

a1 Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK

a2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK

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.

(Received October 18 2011)

(Accepted February 21 2012)

Correspondence:

c1 Corresponding author. E-mail: alison.mcdonald@strath.ac.uk