Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams

We establish the advantages of two-color excitation (2CE) fluorescence microscopy where the two confocal excitation beams are separated by an angle, theta [Opt. Lett. 24 (1999) 1505]. The two excitation wavelengths lambda(1) and lambda(2) are related according to 1/lambda(e) = 1/lambda(1) + 1/lambda(2), where lambda(e) is the single-photon excitation wavelength of the sample. Both circular and annular pupils are considered in the calculation of the three-dimensional point spread function of the 2CE microscope. When lambda(1) not equal lambda(2), the 2CE fluorescence intensity is proportional to the product of the two excitation intensities and is generated only in regions where the excitation beams overlap with each other in both space and time. Two-photon excitation fluorescence (2PE) microscopy is a special case of 2CE microscopy with lambda(1) = lambda(2) = 2 lambda(e) = lambda(2p) With focusing lenses of low-to-moderate numerical apertures, the main advantage of 2CE microscopy over its 2PE counterpart is not in improved imaging resolution, but in the observation of microscopic objects through highly scattering media. In 2CE imaging, scattering decreases the in-focus fluorescence but only minimally increases the unwanted fluorescence background unlike in the 2PE case. Furthermore, annular lenses with their very long depths of field offer an easier way for maintaining the alignment of the two confocal excitation beams in applications involving the imaging of irregularly shaped, thick samples. (C) 2000 Elsevier Science B.V. All rights reserved.