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). We have recently demonstrated that a multianode photomultiplier tube (MAPMT) placed in a descanned configuration can effectively collect scattered emission photons from each focus into their corresponding anodes significantly improving image SNR for highly scattering specimens. Unfortunately, a descanned MMM has a longer detection path resulting in substantial emission photon loss. Optical design constraints in a descanned geometry further results in significant optical aberrations especially for large field-of-view (FOV), high NA objectives. Here, we introduce a non-descanned MMM based on MAPMT that substantially overcomes most of these drawbacks. We show that we improve signal efficiency up to fourfold with limited image SNR degradation due to scattered emission photons. The excitation foci can also be spaced wider to cover the full FOV of the objective with minimal aberrations. The performance of this system is demonstrated by imaging interneuron morphological structures deep in the brains of living mice.

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. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.

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. Further, the depth resolution of the improved system is dictated by the spatial frequency of the structure light with the potential of attaining depth resolution better than point-scanning two-photon microscopy.

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. Prior theoretical models of temporal focusing processes approximate the contributions of the different spectral components by their mean. This is valid for longer pulses that have narrower spectral bandwidth but results in a systematic deviation when broad spectrum, femtosecond pulses are used. Further, prior model takes the paraxial approximation but since these pulses are focused with high numerical aperture (NA) objectives, the effects of the vectorial nature of light should be considered. In this paper we present a paraxial and a vector theory of temporal focusing that takes into account the finite spread of the spectrum. Using paraxial theory we arrive at an analytical solution to the electric field at the focus for temporally focused wide-field two-photon (TF2p) microscopy as well as in the case of a spectrally chirped input beam. We find that using paraxial theory while accounting for the broad spectral spread gives results almost twice vector theory. Experiment results agree with predictions of the vector theory giving an axial full-width half maximum (FWHM) of 2.1 µmand1.8 µmrespectively as long as spectral spread is taken into account. Using our system parameters, the optical sectioning of the TF2p microscope is found to be 8 µm. The optical transfer function (OTF) of a TF2p microscope is also derived and is found to pass a significantly more limited band of axial frequencies than a point scanning two-photon (2p) microscope or a single photon (1p) confocal microscope.

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. This dipole model is not sufficient to describe the effects of refractive index of a scatterer or to describe scattering at the edges of a scatterer. Taking into account the interaction between dipoles overcomes these short comings of the uncoupled dipole model. Coupled dipole model has been primarily used for linear scattering studies but it can be extended to predict nonlinear scattering. The coupled and uncoupled dipole models have been compared to highlight their differences. Results of nonlinear scattering predicted by coupled dipole model agree well with previously reported experimental results.

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. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods.

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. Furthermore, the merits of such a setup are also discussed in relation to surface plasmon microscopy and single-molecule orientation studies, where the doughnut point spread function can be engineered into a single-lobed point spread function.

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. The oscillations of the Gouy phase shift arise from the strength of the nondominant discontinuity.

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. The ratio of the axial component to the transverse component is also the greatest for the uniform pupil function. On the other hand, the Bessel-Gauss beam benefits the most from the use of an annulus.