An official website of the United States government
Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms across multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish andDrosophilalarvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. To our knowledge, as a novel approach for snapshot tomographic capture, ReFLeCT is a major advance toward bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.
more »
« less
Single-Cellular Dynamic Mechanical Analysis of Live 3d Organoids Under Light-Sheet Fluorescence Microscopy
We have developed a novel microscopic analysis system that combines the functions of light-sheet fluorescence microscopy (LSM) and dynamic mechanical analysis (DMA). We have integrated the three uniquely designed components of (i) a MEMS dynamic compression device with a μ-force sensor, (ii) a high-speed 3D light-sheet scanner and an imager, and (iii) a custom-programmed image-based 3D modeling algorithm. Here, we demonstrate spatially-resolved mechanical characterization of viscoelastic materials under high-resolution 3D fluorescence microscopy for the first time.
more »
« less
- PAR ID:
- 10516285
- Publisher / Repository:
- IEEE
- Date Published:
- Journal Name:
- 2024 IEEE 37th International Conference on Micro Electro Mechanical Systems (MEMS)
- ISSN:
- 2160-1968
- ISBN:
- 979-8-3503-5792-9
- Page Range / eLocation ID:
- 330 to 333
- Format(s):
- Medium: X
- Location:
- Austin, TX, USA
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective lightsheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.more » « less
-
Research of cellular and molecular processes by way of histological methods allows for some insight but comes with a fundamental set of constraints that are challenging to overcome. Traditional histological methods are laborious, as well as severely limiting for in-depth study of developmental processes or disease processes in vivo. In traditional histology, fixing and sectioning tissue necessarily eliminates its dynamic function, while tissue section thickness limits the scope of investigation with conventional imaging tools. Noninvasive in vivo study of tissues and biomarkers is therefore paramount in gaining a fuller understanding of the pathophysiology surrounding conditions like congenital heart disorders. Light-sheet fluorescence microscopy (LSFM) is a powerful and noninvasive optical microscopy tool that can image in vivo tissue function in 4D (3D + time). LSFM boasts benefits such as short pixel dwell time (and therefore minimal photobleaching) while maintaining the ability to image a high dynamic range, as well as deep-tissue optical sectioning. Researchers have been seeking to overcome this problem by developing tissue-clearing techniques to attempt to homogenize the refractive index across the tissue via the removal of light-scattering pigments and lipids.more » « less
-
Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.more » « less
-
Transitions of biological tissues between solid‐like and liquid‐like phases have been of great recent interest. Here, the first successful cell‐by‐cell evaluation of tissue viscoelastic transition is presented. An in situ micro‐mechanical perturbation is applied to a microtissue, and the resulting volumetric deformation is evaluated using 3D light‐sheet microscopy and digital image correlation (DIC), quantifying both solid‐like, well‐aligned displacement and liquid‐like swirling motion between individual cells. The viscoelastic transition of fibroblasts is crucial in fundamental physiological events, such as placentation, cancer dissemination, and wound healing. This study investigates 3D organoid systems modeling maternal‐fetal and tumor‐stroma interfaces, demonstrating established molecular and structural parallels. The analysis visualizes individual cells in stromal‐epithelial interactions and how they collectively alter tissue viscoelastic properties. It also enables in‐silico microdissection, linking single‐cell viscoelasticity with multi‐channel fluorescence. RNAseq analysis of endometrial stromal fibroblasts shows that decidualization activates mechano‐transcriptional regulators, including myocardin‐related transcription factors (MRTFs), associated with increased cellular contractility and actomyosin mobilization. Knocking down MRTFA in cancer‐associated fibroblasts in the tumor‐fibroblast co‐culture 3D model induces significant changes in fibroblast properties, mirroring those observed in the maternal‐fetal interface model, highlighting parallels between placentation and cancer invasion. This analysis confirms existing beliefs and discovers new insights broadly applicable to studying organoids, embryos, tumors, and other tissues.more » « less
