Focusing and imaging in microsphere-based microscopy.

Microsphere-based microscopy systems have garnered lots of recent interest, mainly due to their capacity in focusing light and imaging beyond the diffraction limit. In this paper, we present theoretical foundations for studying the optical performance of such systems by developing a complete theoretical model encompassing the aspects of illumination, sample interaction and imaging/collection. Using this model, we show that surface waves play a significant role in focusing and imaging with the microsphere. We also show that by designing a radially polarized convergent beam, we can focus to a spot smaller than the diffraction limit. By exploiting surface waves, we are able to resolve two dipoles spaced 98 nm apart in simulation using light at a wavelength of 402.292 nm. Using our model, we also explore the effect of beam geometry and polarization on optical resolution and focal spot size, showing that both geometry and polarization greatly affect the shape of the spot.

Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

Analytic method to optimize aperture design in focal modulation microscopy.

Focal modulation microscopy (FMM) has been demonstrated more effective than confocal microscopy for imaging of thick biological tissues. To improve its penetration depth further, we propose a simple analytical method to enlarge the modulation depth, the unique property of FMM directly linked to its signal-to-noise ratio. The modulation depth increases as the excitation intensity of the binary phase aperture status is pushed further away from the focal region of the detection optics, thereby creating a dark region in the focal volume, which we call maximally flat crater (MFC). By direct algebraic manipulation, MFCs are achieved for both scalar and vector diffraction optics. Numerical results show that the modulation depth from MFC is very close to the maximum values, with a small difference less than 3% for the same number of subapertures. Applications of bifocus produced by MFC apertures are also discussed.

Phase-preserved optical elevator.

The unique superiority of transformation optics devices designed from coordinate transformation is their capability of recovering both ray trajectory and optical path length in light manipulation. However, very few experiments have been done so far to verify this dual-recovery property from viewpoints of both ray trajectory and optical path length simultaneously. The experimental difficulties arise from the fact that most previous optical transformation optics devices only work at the nano-scale; the lack of intercomparison between data from both optical path length and ray trajectory measurement in these experiments obscured the fact that the ray path was subject to a subwavelength lateral shift that was otherwise not easily perceivable and, instead, was pointed out theoretically [B. Zhang et al. Phys. Rev. Lett. 104, 233903, 2010]. Here, we use a simple macroscopic transformation optics device of phase-preserved optical elevator, which is a typical birefringent optical phenomenon that can virtually lift an optical image by a macroscopic distance, to demonstrate decisively the unique optical path length preservation property of transformation optics. The recovery of ray trajectory is first determined with no lateral shift in the reflected ray. The phase preservation is then verified with incoherent white-light interferometry without ambiguity and phase unwrapping.

Classical imaging theory of a microlens with super-resolution.

Super-resolution in imaging through a transparent spherical microlens has attracted lots of attention because of recent promising experimental results with remarkable resolution improvement. To provide physical insight for this super-resolution phenomenon, previous studies adopted a phenomenological explanation mainly based on the super-focusing effect of a photonic nanojet, while a direct imaging calculation with classical imaging theory has rarely been studied. Here we theoretically model the imaging process through a microlens with vectorial electromagnetic analysis, and then exclude the previously plausible explanation of super-resolution based on the super-focusing effect. The results showed that, in the context of classical imaging theory subject to the two-point resolution criterion, a microlens with a perfect spherical shape cannot achieve the experimentally verified sub-100 nm resolution. Therefore, there must be some other physical mechanisms that contribute to the reported ultrahigh resolution but have not been revealed in theory.

Optical breast atlas as a testbed for image reconstruction in optical mammography.

We present two optical breast atlases for optical mammography, aiming to advance the image reconstruction research by providing a common platform to test advanced image reconstruction algorithms. Each atlas consists of five individual breast models. The first atlas provides breast vasculature surface models, which are derived from human breast dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data using image segmentation. A finite element-based method is used to deform the breast vasculature models from their natural shapes to generate the second atlas, compressed breast models. Breast compression is typically done in X-ray mammography but also necessary for some optical mammography systems. Technical validation is presented to demonstrate how the atlases can be used to study the image reconstruction algorithms. Optical measurements are generated numerically with compressed breast models and a predefined configuration of light sources and photodetectors. The simulated data is fed into three standard image reconstruction algorithms to reconstruct optical images of the vasculature, which can then be compared with the ground truth to evaluate their performance.

Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo.

Multi-dimensional fluorescence imaging of live animal models demands strong optical sectioning, high spatial resolution, fast image acquisition, and minimal photobleaching. While conventional laser scanning microscopes are capable of deep penetration and sub-cellular resolution, they are generally too slow and causing excessive photobleaching for volumetric or time-lapse imaging. We demonstrate the performance of an augmented line-scan focal modulation microscope (aLSFMM), a high-speed imaging platform that affords above video-rate imaging speed by the use of line scanning. Exceptional background rejection is accomplished by combining a confocal slit with focal modulation. The image quality is further improved by merging the information from simultaneously acquired focal modulation and confocal images. Such a hybrid imaging scheme makes it possible to use very low power excitation light in high-speed imaging, and therefore leads to reduced photobleaching that is desirable for three-dimensional (3D) and four-dimensional (4D) in vivo image acquisition.