Effect of a confocal pinhole in two-photon microscopy.
We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.
Gauderon
R
R
Physical Optics Department, School of Physics A28, University of Sydney, NSW 2006, Australia.
Lukins
P B
PB
Sheppard
C J
CJ
D000535:Aluminum; D001881:Borates / Q000737:chemistry; D002736:Chloroplasts; D002875:Chromosomes; D007089:Image Enhancement; D018020:Lithium Compounds / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D019697:Onions / Q000166:cytology; D017785:Photons; D018517:Plant Roots / Q000648:ultrastructure; D018724:Spinacia oleracea / Q000166:cytology
D001881:Borates; D018020:Lithium Compounds; D000535:Aluminum; C021051:lithium borate