Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

Strategic and practical guidelines for successful structured illumination microscopy

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d

ENHANCEMENTS TO THE MODIFIED COMPOSITE PATTERN METHOD OF STRUCTURED LIGHT 3D CAPTURE

The use of structured light illumination techniques for three-dimensional data acquisition is, in many cases, limited to stationary subjects due to the multiple pattern projections needed for depth analysis. Traditional Composite Pattern (CP) multiplexing utilizes sinusoidal modulation of individual projection patterns to allow numerous patterns to be combined into a single image. However, due to demodulation artifacts, it is often difficult to accurately recover the subject surface contour information. On the other hand, if one were to project an image consisting of many thin, identical stripes onto the surface, one could, by isolating each stripe center, recreate a very accurate representation of surface contour. But in this case, recovery of depth information via triangulation would be quite difficult. The method described herein, Modified Composite Pattern (MCP), is a conjunction of these two concepts. Combining a traditional Composite Pattern multiplexed projection image with a pattern of thin stripes allows for accurate surface representation combined with non-ambiguous identification of projection pattern elements. In this way, it is possible to recover surface depth characteristics using only a single structured light projection. The technique described utilizes a binary structured light projection sequence (consisting of four unique images) modulated according to Composite Pattern methodology. A stripe pattern overlay is then applied to the pattern. Upon projection and imaging of the subject surface, the stripe pattern is isolated, and the composite pattern information demodulated and recovered, allowing for 3D surface representation. In this research, the MCP technique is considered specifically in the context of a Hidden Markov Process Model. Updated processing methodologies explained herein make use of the Viterbi algorithm for the purpose of optimal analysis of MCP encoded images. Additionally, we techniques are introduced which, when implemented, allow fully automated processing of the Modified Composite Pattern image

A Coded Structured Light System Based on Primary Color Stripe Projection and Monochrome Imaging

Coded Structured Light techniques represent one of the most attractive research areas within the field of optical metrology. The coding procedures are typically based on projecting either a single pattern or a temporal sequence of patterns to provide 3D surface data. In this context, multi-slit or stripe colored patterns may be used with the aim of reducing the number of projected images. However, color imaging sensors require the use of calibration procedures to address crosstalk effects between different channels and to reduce the chromatic aberrations. In this paper, a Coded Structured Light system has been developed by integrating a color stripe projector and a monochrome camera. A discrete coding method, which combines spatial and temporal information, is generated by sequentially projecting and acquiring a small set of fringe patterns. The method allows the concurrent measurement of geometrical and chromatic data by exploiting the benefits of using a monochrome camera. The proposed methodology has been validated by measuring nominal primitive geometries and free-form shapes. The experimental results have been compared with those obtained by using a time-multiplexing gray code strategy

Optical techniques for 3D surface reconstruction in computer-assisted laparoscopic surgery

One of the main challenges for computer-assisted surgery (CAS) is to determine the intra-opera- tive morphology and motion of soft-tissues. This information is prerequisite to the registration of multi-modal patient-specific data for enhancing the surgeon’s navigation capabilites by observ- ing beyond exposed tissue surfaces and for providing intelligent control of robotic-assisted in- struments. In minimally invasive surgery (MIS), optical techniques are an increasingly attractive approach for in vivo 3D reconstruction of the soft-tissue surface geometry. This paper reviews the state-of-the-art methods for optical intra-operative 3D reconstruction in laparoscopic surgery and discusses the technical challenges and future perspectives towards clinical translation. With the recent paradigm shift of surgical practice towards MIS and new developments in 3D opti- cal imaging, this is a timely discussion about technologies that could facilitate complex CAS procedures in dynamic and deformable anatomical regions