Selective Plane Illumination Microscopy System Research Articles

Integrated light-sheet illumination using metallic slit microlenses.

Light sheet microscopy (LSM) - also known as selective plane illumination microscopy (SPIM) - enables high-speed, volumetric imaging by illuminating a two-dimensional cross-section of a specimen. Typically, this light sheet is created by table-top optics, which limits the ability to miniaturize the overall SPIM system. Replacing this table-top illumination system with miniature, integrated devices would reduce the cost and footprint of SPIM systems. One important element for a miniature SPIM system is a flat, easily manufactured lens that can form a light sheet. Here we investigate planar metallic lenses as the beam shaping element of an integrated SPIM illuminator. Based on finite difference time domain (FDTD) simulations, we find that diffraction from a single slit can create planar illumination with a higher light throughput than zone plate or plasmonic lenses. Metallic slit microlenses also show broadband operation across the entire visible range and are nearly polarization insensitive. Furthermore, compared to meta-lenses based on sub-wavelength-scale diffractive elements, metallic slit lenses have micron-scale features compatible with low-cost photolithographic manufacturing. These features allow us to create inexpensive integrated devices that generate light-sheet illumination comparable to tabletop microscopy systems. Further miniaturization of this type of integrated SPIM illuminators will open new avenues for flat, implantable photonic devices for in vivo biological imaging.

Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

High-NA open-top selective-plane illumination microscopy for biological imaging

Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for the volumetric imaging of living organisms over extended times. However, the spatial configuration of a SPIM system often limits its compatibility with many widely used biological sample holders such as multi-well chambers and plates. To solve this problem, we developed a high numerical aperture (NA) open-top configuration that places both the excitation and detection objectives on the opposite of the sample coverglass. We carried out a theoretical calculation to analyze the structure of the system-induced aberrations. We then experimentally compensated the system aberrations using adaptive optics combined with static optical components, demonstrating near-diffraction-limited performance in imaging fluorescently labeled cells.

A dual-modality optical coherence tomography and selective plane illumination microscopy system for mouse embryonic imaging.

Both optical coherence tomography (OCT) and selective plane illumination microscopy (SPIM) are frequently used in mouse embryonic research for high-resolution three-dimensional imaging. Each of these imaging methods provide a unique and independent advantage: SPIM provides morpho-functional information through immunofluorescence and OCT provides a method for whole-embryo 3D imaging. In this study, we have combined rotational imaging OCT and SPIM into a single, dual-modality device to image E9.5 mouse embryos. The results demonstrate that the dual-modality setup is able to provide both anatomical and functional information simultaneously for more comprehensive tissue characterization.

Advancing ovarian folliculometry with selective plane illumination microscopy.

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. The large field of view and fast acquisition speed of our SPIM system enables rendering of volumetric image stacks from intact whole porcine ovarian follicles, clearly visualizing follicular features including follicle volume and average diameter (70 μm–2.5 mm), their spherical asymmetry parameters, size of developing cumulus oophorus complexes (40 μm–110 μm), and follicular wall thickness (90 μm–120 μm). Follicles at all developmental stages were identified. A distribution of the theca thickness was measured for each follicle, and a relationship between these distributions and the stages of follicular development was discerned. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

Fluorescence Anisotropy Imaging in 3D with Single Plane Illumination Microscopy

Anisotropy imaging is a powerful tool to study the organization, composition and dynamics of molecules in biological systems. Changes in the organization and conformation of biomolecules within cells and tissues can be visualized at the single pixel level. Fluorescence anisotropy is typically probed with laser scanning and epifluorescence-based techniques. However, those techniques are limited in either axial resolution, image acquisition speed, or, by photobleaching. In the last decade, selective plane illumination microscopy (SPIM) has emerged as the best choice for three-dimensional time lapse imaging since it combines axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure [1,2]. We demonstrate how SPIM can be used for three-dimensional fluorescence anisotropy imaging of live cells [3]. To verify the accuracy of our SPIM system, we first measured and compared the anisotropy of Rhodamine110 solutions with different water/glycerol ratios with results obtained using a spectrofluorimeter. Then, we visualized the aggregation states of different fusion proteins in live cells. Further, we were able to show by 3D time lapse anisotropy imaging that formation of membrane protrusions and paxillin aggregates precedes focal adhesion assembly.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.[1] Huisken, J. et al., Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007-1009 (2004).[2] Hedde, P.N. et al., Rapid Measurement of Molecular Transport and Interaction inside Living Cells Using Single Plane Illumination. Sci. Rep. 4, 7048 (2014).[3] Hedde, P.N. et al., 3D fluorescence anisotropy imaging using selective plane illumination microscopy. Opt. Express 23, 22308-22317 (2015).