Ke Si

Improved spatial resolution in fluorescence focal modulation microscopy

We show that the focal modulation microscopy (FMM), which combines a spatial phase modulator with confocal microscopy, results in an improvement in spatial resolution. This technique was introduced to increase imaging depth into tissue and rejection of background from a thick scattering object. A theory for image formation in FMM is presented, and the effects of detecting the in-phase modulated fluorescence signal are discussed. Compared with conventional confocal microscopy, the width of the point-spread function for the in-phase fluorescence signal is improved by 16.4%. When applied to saturable fluorescence, the half-width at half-maximum is improved by 33.6%, 50.0%, and 62.9%, at demodulation frequencies 2omega, 4omega, and 8omega, respectively.

Optimization of axial resolution in a confocal microscope with D-shaped apertures

We show theoretically that the axial resolution is improved when two centrosymmetric D-shaped apertures are combined in a confocal microscope with a finite-sized pinhole. The optimum width of a divider that separates the D-shaped apertures to give the maximum axial resolution for a given pinhole size is investigated, and the magnitude of the signal level is explored.

The divided aperture technique for microscopy through scattering media

A diffraction analysis is presented for image formation in confocal microscopy using the divided aperture technique, which uses two D-shaped apertures (also called specular microscopy). The effects of increasing the width of a divider, that separates the two D shapes, are investigated. As the width is increased, the resolution degrades. The efficiency of singly-scattered light rejection is not improved with increased width.

Three-dimensional coherent transfer function for a confocal microscope with two D-shaped pupils

The three-dimensional coherent transfer function for D-shaped pupils in reflection-mode confocal scanning microscopy is analytically derived under the paraxial approximation. Three-dimensional numerical plots are presented, showing the dependence of the transfer functions on the width of a central divider. The applications in fiber-optical confocal scanning microscopy are also discussed.

Focal modulation microscopy with annular apertures: A numerical study

Image formation of focal modulation microscopy with annular aperture (AFMM) is presented. The spatial resolution is discussed. Compared with confocal microscopy, AFMM can simultaneously enhance the axial and transverse resolution. By adjusting the width of the annular objective aperture, AFMM can be adjusted from best spatial resolution performance to highest signal level. The capability of background rejection is also investigated, showing AFMM has the potential to further increase the imaging penetration depth. Finally, an optimal configuration of AFMM is elaborated.

Polarization effects in 4Pi Microscopy

The effects of different apodization conditions and polarization distributions on imaging in 4Pi microscopy are discussed. Performance parameters are derived that allow the different implementations to be compared. 4Pi microscopy is mainly used because of its superior axial imaging performance, but it is shown that transverse resolution is also improved in the 4Pi geometry, by as much as 25% compared with focusing by a single aplanatic lens. Compared with plane-polarized illumination in a 4Pi aplanatic system, transverse resolution in the 4Pi mode can also be increased by about 18%, using radially polarized illumination, but at the expense of axial resolution. The electric energy density at the focus for a given power input can be increased using electric dipole polarization, which is relevant for atomic physics experiments such as laser trapping and cooling.

Two-photon focal modulation microscopy in turbid media

The image formation of two-photon focal modulation microscopy (2PFMM) in turbid media is theoretically investigated. The results show that compared with conventional two-photon fluorescence microscopy, the ballistic excitation of 2PFMM is concentrated in a much smaller region around the focal point and decays more rapidly outside the focal volume, while the scattered excitation is largely suppressed. When focuses at 1600 μm, the signal-to-background ratio and signal-to-noise ratio of 2PFMM are improved by 30 dB and 18 dB, respectively, indicating that 2PFMM can achieve a large imaging penetration depth.

Divided-aperture technique for fluorescence confocal microscopy through scattering media

We present a diffraction analysis for image formation in fluorescence confocal microscopy with divided apertures. The three-dimensional optical transform function is given, and axial resolution and transverse resolution are investigated. The results show that the employment of a divided-aperture technique in fluorescence confocal microscopy can enhance the rejection of background scattering. In addition, the axial resolution and transverse resolution can be improved by adjusting the width of the divider strip. For a given detector size, an optimum value of the divider strip width is given to obtain the best axial resolution or transverse resolution. The integrated intensity and signal-to-background ratio are also discussed.

Edge enhancement for in-phase focal modulation microscope

In-phase focal modulation microscopy (IPFMM) with single photon excited fluorescence is presented. Optical transfer functions and images of thin and thick fluorescent edges in IPFMM are investigated. The results show that, compared with confocal microscopy, using IPFMM can result in a sharper image of the edge, and the edge gradient can be increased up to 75.4% and 58.9% for a thick edge and a thin edge, respectively. Signal level is also discussed, and the results show that, to obtain high transverse resolution with IPFMM, the normalized detector pinhole radius should not exceed 2.8.

Model for light scattering in biological tissue and cells based on random rough nonspherical particles

A random nonspherical model for biological tissue and cells permits a better description of their optical properties. Rough surface nonspherical particles have been employed to model biological tissue and cells. The phase function, the anisotropy factor of scattering, and the reduced scattering coefficient are derived. The effect of different size distributions is also discussed. The theoretical results show good agreement with experimental data.