3D Microscopy using Confocal Microscopy
The importance of confocal fluorescence microscopy in modem biological research results from its optical sectioning capability, which allows the three-dimensional analysis of thick specimens. This property is due to the combination of a point-like light source and a point-like detector, which restrict the illumination and detection volumes, respectively. Only the volume that is illuminated and detected is relevant to the confocal observation volume. The smaller it is, the better is the resolution. The performance of a confocal microscope is thus primarily specified by the spatial extent of the confocal point spread function (PSF). The extent can be estimated, e.g., by the volume enclosed by the isosurface at half maximum of the PSF (VHM – volume at half maximum).The relationship of the parameters that determine the lateral resolution of a microscope has been described by Ernst Abbé. The diameter of a light spot in the focal plane Δx is proportional to the wavelength λ of the incident light and inversely proportional to the numerical aperture of the optical system (N.A. = n, ∙ sin α).