Enhancement of image quality in planar Airy light-sheet microscopy via subtraction method

The use of propagation-invariant Airy beams enables a light-sheet microscopy with a large field-of-view. Without relying upon two-photon excitation or deconvolution-based processing to eliminate out-of focus blur caused by the side lobes, here, we present how the subtraction method is applied to enhance the image quality in digital scanned light-sheet microscopy with Airy beam. In the proposed method, planar Airy beam with the symmetric transversal structure is used to excite the sample. A hollow Airy beam with zero intensity at the focal plane is created, which is mainly used to excite the out-of-focus signal. By scanning the sample twice with the normal planar Airy beam and the hollow Airy beam, digital post-processing of the obtained images by subtraction allows for the rejection of out-of-focus blur and improves the optical sectioning, the axial resolution and the intensity distribution uniformity of the light-sheet microscopy.

Digital Image Analysis Tools Developed by the Indiana O’Brien Center

The scale and complexity of images collected in biological microscopy have grown enormously over the past 30 years. The development and commercialization of multiphoton microscopy has promoted a renaissance of intravital microscopy, providing a window into cell biology in vivo. New methods of optical sectioning and tissue clearing now enable biologists to characterize entire organs at subcellular resolution. New methods of multiplexed imaging support simultaneous localization of forty or more probes at a time. Exploiting these exciting new techniques has increasingly required biomedical researchers to master procedures of image analysis that were once the specialized province of imaging experts. A primary goal of the Indiana O’Brien Center has been to develop robust and accessible image analysis tools for biomedical researchers. Here we describe biomedical image analysis software developed by the Indiana O’Brien Center over the past 25 years.

Whole-Brain Reconstruction of Neurons in the Ventral Pallidum Reveals Diverse Projection Patterns

The ventral pallidum (VP) integrates reward signals to regulate cognitive, emotional, and motor processes associated with motivational salience. Previous studies have revealed that the VP projects axons to many cortical and subcortical structures. However, descriptions of the neuronal morphologies and projection patterns of the VP neurons at the single neuron level are lacking, thus hindering the understanding of the wiring diagram of the VP. In this study, we used recently developed progress in robust sparse labeling and fluorescence micro-optical sectioning tomography imaging system (fMOST) to label mediodorsal thalamus-projecting neurons in the VP and obtain high-resolution whole-brain imaging data. Based on these data, we reconstructed VP neurons and classified them into three types according to their fiber projection patterns. We systematically compared the axonal density in various downstream centers and analyzed the soma distribution and dendritic morphologies of the various subtypes at the single neuron level. Our study thus provides a detailed characterization of the morphological features of VP neurons, laying a foundation for exploring the neural circuit organization underlying the important behavioral functions of VP.

Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons

Multiphoton (MP) microscopy is a well-established method for the non-invasive imaging of biological tissues. However, its optical sectioning capabilities are reduced due to specimen-induced aberrations. Both the manipulation of spherical aberration (SA) and the use of axicons have been reported to be useful techniques to bypass this limitation. We propose the combination of SA patterns and variable axicons to further improve the quality of MP microscopy images. This approach provides enhanced images at different depth locations whose quality is better than those corresponding to the use of SA or axicons separately. Thus, the procedure proposed herein facilitates the visualization of details and increases the depth observable at high resolution.

Miniature Structured Illumination Microscope for in vivo 3D Imaging of Brain Structures with Optical Sectioning

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 μm. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 220 μm is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 μm in the brain.

Acceleration-Aware Path Planning with Waypoints

In this article we demonstrate that acceleration and deceleration of direction-turning drones at waypoints have a significant influence to path planning which is important to be considered for time-critical applications, such as drone-supported search and rescue. We present a new path planning approach that takes acceleration and deceleration into account. It follows a local gradient ascend strategy which locally minimizes turns while maximizing search probability accumulation. Our approach outperforms classic coverage-based path planning algorithms, such as spiral- and grid-search, as well as potential field methods that consider search probability distributions. We apply this method in the context of autonomous search and rescue drones and in combination with a novel synthetic aperture imaging technique, called Airborne Optical Sectioning (AOS), which removes occlusion of vegetation and forest in real-time.

Comparison of Two- and Three-Beam Interference Pattern Generation in Structured Illumination Microscopy

Structured illumination microscopy (SIM) provides wide-field optical sectioning in the focal plane by modulating the imaging information using fringe pattern illumination. For generating the fringe pattern illumination, a digital micro-mirror device (DMD) is commonly used due to its flexibility and fast refresh rate. However, the benefit of different pattern generation, for example, the two-beam interference mode and the three-beam interference mode, has not been clearly investigated. In this study, we systematically analyze the optical sectioning provided by the two-beam inference mode and the three-beam interference mode of DMD. The theoretical analysis and imaging results show that the two-beam interference mode is suitable for fast imaging of the superficial dynamic target due to reduced number of phase shifts needed to form the image, and the three-beam interference mode is ideal for imaging three-dimensional volume due to its superior optical sectioning by the improved modulation of the illumination patterns. These results, we believe, will provide better guidance for the use of DMD for SIM imaging and also for the choice of beam patterns in SIM application in the future.

A single molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters and optical aberrations. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organisation and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

Computational optical sectioning with an incoherent multiscale scattering model for light-field microscopy

Quantitative volumetric fluorescence imaging at high speed across a long term is vital to understand various cellular and subcellular behaviors in living organisms. Light-field microscopy provides a compact computational solution by imaging the entire volume in a tomographic way, while facing severe degradation in scattering tissue or densely-labelled samples. To address this problem, we propose an incoherent multiscale scattering model in a complete space for quantitative 3D reconstruction in complicated environments, which is called computational optical sectioning. Without the requirement of any hardware modifications, our method can be generally applied to different light-field schemes with reduction in background fluorescence, reconstruction artifacts, and computational costs, facilitating more practical applications of LFM in a broad community. We validate the superior performance by imaging various biological dynamics in Drosophila embryos, zebrafish larvae, and mice.