Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes.
Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.
Heintzmann
Rainer
R
Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. rheintz@gwdg.de
Sarafis
Vassilios
V
Munroe
Paul
P
Nailon
John
J
Hanley
Quentin S
QS
Jovin
Thomas M
TM
D016475:3T3 Cells / Q000648:ultrastructure; D000818:Animals; D019068:Bryopsida / Q000648:ultrastructure; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D013170:Spores / Q000648:ultrastructure; D013382:Subtraction Technique