Wavelength Fluorescence Confocal Microscopy Imaging Characteristics

Two-photon confocal microscopy has attracted more and more attention recently, as well as fluorescent confocal scanning microscopy. Due to its supper-resolution imaging ability and its unique three-dimensional microfabrication ability. It has been widely used in life science , 3D-optical data storage , and lithographic microfabrication . The imaging principles of confocal microscopy have been discussed extensively by many authors , but all of those theories didn't account for the nonlinear effect of two-photon excitation. Because of the nonlinear effect, the imaging property of two-photon confocal microscopy is very different from that of the conventional confocal microscopy.That the fluorescent wavelength equals to the excitation wavelength has been discussed theoretically as we know. So these imaging theories cannot describe the imaging property of confocal scanning microscopy precisely.Fluorescence power transfer function, three-dimensional point spread function(3D-PSF) and three-dimensional optical transfer function(SD-OTF) for the various fluorescent wavelength of the two kinds of fluorescence confocal scanning microscopy are calculated in this paper by using Fourier imaging theory. The results show that the fluorescent wavelength has influence on imaging property of confocal microscopy such as spatial cut-off frequency, resolution and 3D-OTF. There is a different missing-cone in the 3-D space of OTF when the ratio of excitation wavelength to fluorescent wavelength decreases.Finally, a confocal scanning microscopy, designed and made by us, is introduced, the images of some sample are provided.