Non-descanned multifocal multiphoton microscopy with a multianode photomultiplier tube.

Multifocal multiphoton microscopy (MMM) improves imaging speed over a point scanning approach by parallelizing the excitation process. Early versions of MMM relied on imaging detectors to record emission signals from multiple foci simultaneously. For many turbid biological specimens, the scattering of emission photons results in blurred images and degrades the signal-to-noise ratio (SNR).

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy.

Optical sectioning techniques offer the ability to acquire three-dimensional information from various organ tissues by discriminating between the desired in-focus and out-of-focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.

High-throughput nonlinear optical microscopy.

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics.

Parallel super-resolution imaging.

Massive parallelization of scanning-based super-resolution imaging allows fast imaging of large fields of view.

Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination.

Although temporally focused wide-field two-photon microscopy (TFM) can perform depth resolved wide field imaging, it cannot avoid the image degradation due to scattering of excitation and emission photons when imaging in a turbid medium. Further, its axial resolution is inferior to standard point-scanning two-photon microscopy. We implemented a structured light illumination for TFM and have shown that it can effectively reject the out-of-focus scattered emission photons improving image contrast.

Temporally focused wide-field two-photon microscopy: paraxial to vectorial.

Temporal focusing allows for optically sectioned wide-field microscopy. The optical sectioning arises because this method takes a pulsed input beam, stretches the pulses by diffracting off a grating, and focuses the stretched pulses such that only at the focal plane are the pulses re-compressed. This approach generates nonlinear optical processes at the focal plane and results in depth discrimination.

Coupled and uncoupled dipole models of nonlinear scattering.

Dipole models are one of the simplest numerical models to understand nonlinear scattering. Existing dipole model for second harmonic generation, third harmonic generation and coherent anti-Stokes Raman scattering assume that the dipoles which make up a scatterer do not interact with one another. Thus, this dipole model can be called the uncoupled dipole model.

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment.

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm.

Polarization conversion in confocal microscopy with radially polarized illumination.

The effects of using radially polarized illumination in a confocal microscope are discussed, and the introduction of a polarization mode converter into the detection optics of the microscope is proposed. We find that with such a configuration, bright-field imaging can be performed without losing the resolution advantage of radially polarized illumination. The detection efficiency can be increased by three times without having to increase the pinhole radius and sacrificing the confocality of the system.

Fractional Gouy phase.

In general, the total Gouy phase shift has the form n pi, where n need not be an integer. As a result of the Fourier transforming property of a lens, the Gouy phase is found to be related to the types of discontinuities at the upper or lower range of the pupil function Q(c) resulting from the asymptotic order of the Fourier transform. The sign of the Gouy phase is also related to the slope of the pupil function.

Performance parameters for focusing of radial polarization.

Performance parameters are presented for high-aperture radially polarized focusing systems. These can be used for comparing the focusing performance of different optical systems, including the effect of pupil filters.

Tight focusing of radially polarized Gaussian and Bessel-Gauss beams.

We examine the effects of tightly focusing a radially polarized beam with uniform, Gaussian, or Bessel-Gauss pupil functions. The resulting FWHM is smallest for the case of a uniform amplitude profile, while the Bessel-Gauss beam results in the largest FWHM. The uniform amplitude profile also results in an axial field component that increases fastest with increasing NA.