Primary spherical aberration in two-color (two-photon) excitation fluorescence microscopy with two confocal excitation beams
ABSTRACT We study the effects of primary spherical aberration on the three-dimensional point spread function (PSF) of the two-color (two-photon) excitation (2CE) (2PE) fluorescence microscope with two confocal excitation beams that are separated by an angle theta. The two excitation wavelengths lambda1 and lambda2 are related to the single-photon excitation wavelength lambda(e) by: 1/lambda(e) = 1/lambda1 + 1/lambda2. The general case is considered where both focused beams independently suffer from spherical aberration. For theta = 0, pi/2, and pi, the resulting deterioration of the PSF structure is evaluated for different values of the spherical aberration coefficients via the Linfoot's criteria of fidelity, structural content, and correlation quality. The corresponding degradation of the peak 2CE fluorescence intensity is also determined. Our findings are compared with that of the 2PE fluorescence (lambda1 = lambda2) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the pi/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.
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ABSTRACT: We study the effect of a kind of binary phase-only filters, the Toraldo filters, in two-color excitation fluorescence microscopy. We show that by simple insertion of a properly designed Toraldo filter in one of the illumination arms the axial resolution of the system is significantly improved. Specifically, the main peak of the point spread function is narrowed by 22% along the axial direction.Optics Communications 02/2005; 4230(4-6). DOI:10.1016/j.optcom.2004.11.035 · 1.54 Impact Factor
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ABSTRACT: Optical-beam induced current (OBIC) is generated in blue light-emitting diode samples (peak emission wavelength=480 nm) via two-color (two-photon) excitation using the second- (502.9 nm) and third-order (635.9 nm) Stokes beam outputs of a hydrogen Raman shifter that is pumped by the 355 nm pulsed output (5.4 ns pulsewidth) of a 10 Hz Nd:YAG laser. Two-color OBIC generation happens only in sample regions where the two focused Stokes beams overlap in both space and time. We have verified the direct proportionality of the two-color OBIC signal with the product of the individual Stokes beam intensities as well as the localized nature of two-color OBIC generation in the common focal volume of the two confocal excitation beams.Optics Communications 02/2005; 246(1):117-122. DOI:10.1016/j.optcom.2004.10.059 · 1.54 Impact Factor
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ABSTRACT: We analyze the influence of residual two-photon excitation (2PE) in two-color (two-photon) optical beam induced current (2CE-OBIC) generation in wide band gap semiconductor samples. 2CE-OBIC generation is accomplished with two confocal excitation beams of separation angle θ and wavelengths λ1 and λ2 where λe-1=λ1-1+λ2-1, λe=hc/Eb, h is the Planck’s constant, c is speed of light in vacuum, and Eb is the energy band gap. Because the conduction band of the sample is a continuum, at least one excitation beam would also contribute an undesirable 2PE-OBIC signal that degrades the signal-to-noise ratio of the measured 2CE-OBIC response and broadens the effective OBIC distribution in the sample particularly when θ≠0 or π. We show that the deleterious effects of crosstalk are reduced by a careful selection of λ1 and λ2 and the relative excitation beam intensities. λ1 and λ2 should be chosen to minimize the ratio of the two-photon absorption coefficients (β1,β2) to the 2CE absorption coefficient β12 or at least satisfy the constraint: β1+β2⩽β12. Keeping the two excitation intensities equal is beneficial only when β1=β2. Otherwise, it is advantageous to bias the intensity ratio towards the wavelength with a lower 2PE absorption coefficient.Optics Communications 02/2007; 270(2):139-144. DOI:10.1016/j.optcom.2006.09.051 · 1.54 Impact Factor