Automatic motion compensation for structured illumination endomicroscopy using a flexible fiber bundle

Significance: Confocal laser scanning enables optical sectioning in clinical fiber bundle endomicroscopes, but lower-cost, simplified endomicroscopes use widefield incoherent illumination instead. Optical sectioning can be introduced in these simple systems using structured illumination microscopy (SIM), a multiframe digital subtraction process. However, SIM results in artifacts when the probe is in motion, making the technique difficult to use in vivo and preventing the use of mosaicking to synthesize a larger effective field of view (FOV). Aim: We report and validate an automatic motion compensation technique to overcome motion artifacts and allow generation of mosaics in SIM endomicroscopy. Approach: Motion compensation is achieved using image registration and real-time pattern orientation correction via a digital micromirror device. We quantify the similarity of moving probe reconstructions to those acquired with a stationary probe using the relative mean of the absolute differences (MAD). We further demonstrate mosaicking with a moving probe in mechanical and freehand operation. Results: Reconstructed SIM images show an improvement in the MAD from 0.85 to 0.13 for lens paper and from 0.27 to 0.12 for bovine tissue. Mosaics also show vastly reduced artifacts. Conclusion: The reduction in motion artifacts in individual SIM reconstructions leads to mosaics that more faithfully represent the morphology of tissue, giving clinicians a larger effective FOV than the probe itself can provide

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

Computational Optical Sectioning in Fibre Bundle Endomicroscopy

The field of fibre bundle endomicroscopy has emerged to enable real-time imaging of cellular level features in-vivo. The gold standard is confocal laserscanning, enabling optical sectioning. Point-scan confocal suffers from lower speeds, a need for complex alignment, and the added cost of a laser. This thesis presents three developments in computational optical sectioning for fibre bundle endomicroscopy.The first development is in structured illumination (SIM) endomicroscopy. Lower-cost, simplified endomicroscopes have been developed which use widefield incoherent illumination. Optical sectioning can be introduced to these systems using SIM. SIM improves imaging using spatial modulation of the focal plane and capturing a three-frame sequence. The acquired images are then numerically processed to reject out-of-focus light. This thesis reports and characterises the first high-speed SIM endomicroscope built using a miniature array ofmirrors, a digital micromirror device. The second development is automated motion compensation in SIM endomicroscopy. As a multi frame process, SIM is susceptible to motion artefacts, making the technique difficult to use in vivo and preventing the use of mosaicking to synthesise a larger effective field of view. I report and validate an automatic motion compensation technique to overcome motion artefacts and report the firstmosaics in SIM endomicroscopy.The third development is improvements in subtraction-based enhanced line scanning (ELS) endomicroscopy. The 2D scanning of a point scan confocal endomicroscope can be replaced by a scanning line which is synchronised to the sequential readout of a rolling shutter camera. While this leads to high-speed sectioning, as with all line scanning systems, far-from-focus light degrades images. It is possible to remove this by subtracting a second image taken with an offset detection slit. This has previously required two-cameras or two sequentialframes. The latter introduces motion artefacts. This thesis presents a novel approach to ELS using single frame acquisition with real-time mosaicking at 240frames/s

Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses

Genetically encoded calcium indicators for visualizing dynamic cellular activity have greatly expanded our understanding of the brain. However, due to light scattering properties of the brain as well as the size and rigidity of traditional imaging technology, in vivo calcium imaging has been limited to superficial brain structures during head fixed behavioral tasks. This limitation can now be circumvented by utilizing miniature, integrated microscopes in conjunction with an implantable microendoscopic lens to guide light into and out of the brain, thus permitting optical access to deep brain (or superficial) neural ensembles during naturalistic behaviors. Here, we describe procedural steps to conduct such imaging studies using mice. However, we anticipate the protocol can be easily adapted for use in other small vertebrates. Successful completion of this protocol will permit cellular imaging of neuronal activity and the generation of data sets with sufficient statistical power to correlate neural activity with stimulus presentation, physiological state, and other aspects of complex behavioral tasks. This protocol takes 6–11 weeks to complete

Development and clinical translation of optical and software methods for endomicroscopic imaging

Endomicroscopy is an emerging technology that aims to improve clinical diagnostics by allowing for in vivo microscopy in difficult to reach areas of the body. This is most commonly achieved by using coherent fibre bundles to relay light for illumination and imaging to and from the area under investigation. Endomicroscopy’s attraction for researchers and clinicians is two-fold: on the one hand, its use can reduce the invasiveness of a diagnostic procedure by removing the need for biopsies; On the other hand, it allows for structural and functional in vivo imaging. Endomicroscopic images acquired through optical fibre bundles exhibit artefacts that deteriorate image quality and contrast. This thesis aims to improve an existing endomicroscopy imaging system by exploring two methods that mitigate these artefacts. The first, software-based method takes several processing steps from literature and implements them in an existing endomicroscopy device with a focus on real-time application to enable clinical use, after image quality was found to be inadequate without further processing. A contribution to the field is that two different approaches are implemented and compared in quantitative and qualitative means that have not been compared directly in this manner before. This first attempt at improving endomicroscopy image quality relies solely on digital image processing methods and is developed with a strong focus on real-time applicability in clinical use. Both approaches are compared on pre-clinical and clinical human imaging data. The second method targets the effect of inter-core coupling, which reduces contrast in fibre images. A parallelised confocal imaging method is developed in which a sequence of images is acquired while selectively illuminating groups of fibre cores through the use of a spatial light modulator. A bespoke algorithm creates a composite image in a final processing step. In doing so, unwanted light is detected and removed from the final image. This method is shown to reduce the negative impact of inter-core coupling on image contrast on small imaging targets, while no benefit was found in large, scattering samples

NASA Technology Applications Team: Commercial applications of aerospace technology

The Research Triangle Institute (RTI) is pleased to report the results of NASA contract NASW-4367, 'Operation of a Technology Applications Team'. Through a period of significant change within NASA, the RTI Team has maintained its focus on helping NASA establish partnerships with U.S. industry for dual use development and technology commercialization. Our emphasis has been on outcomes, such as licenses, industry partnerships and commercialization of technologies that are important to NASA in its mission of contributing to the improved competitive position of U.S. industry. RTI's ongoing commitment to quality and customer responsiveness has driven our staff to continuously improve our technology transfer methodologies to meet NASA's requirements. For example, RTI has emphasized the following areas: (1) Methodology For Technology Assessment and Marketing: RTI has developed an implemented effective processes for assessing the commercial potential of NASA technologies. These processes resulted from an RTI study of best practices, hands-on experience, and extensive interaction with the NASA Field Centers to adapt to their specific needs; (2) Effective Marketing Strategies: RTI surveyed industry technology managers to determine effective marketing tools and strategies. The Technology Opportunity Announcement format and content were developed as a result of this industry input. For technologies with a dynamic visual impact, RTI has developed a stand-alone demonstration diskette that was successful in developing industry interest in licensing the technology; and (3) Responsiveness to NASA Requirements: RTI listened to our customer (NASA) and designed our processes to conform with the internal procedures and resources at each NASA Field Center and the direction provided by NASA's Agenda for Change. This report covers the activities of the Research Triangle Institute Technology Applications Team for the period 1 October 1993 through 31 December 1994