Electron microscopic measurement of the size of the optical focus in laser scanning microscopy.

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.