- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Probing Cell Mechanics with Bead-Free Optical Tweezers in the <em>Drosophila</em> Embryo
摘要： Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.
关键词： Drosophila embryo,Developmental Biology,in vivo imaging,optical tweezers,Light sheet microscopy,force measurements,Issue 141,cell mechanics
更新于2025-09-23 15:21:01
-
Analysis of aberrations and performance evaluation of adaptive optics in two-photon light-sheet microscopy
摘要： Two-photon light-sheet microscopy (TP-LSM) system performance is greatly degraded by specimen-induced aberrations in illumination path, which limit the field of view, axial resolution and excitation efficiency of the system. Adaptive optics (AO) is an effective method for attenuating these effects. For the design and evaluation of an AO system, a comprehensive analysis of the effects of aberrations is needed. In this paper, a TP-LSM system is simulated, and new indexes based on integral intensity are introduced for the evaluation of an aberrated light-sheet. Then, the influences of each Zernike mode and random aberrations on the illumination path of the TP-LSM system are investigated with a numerical simulation method. Results show that high-order aberrations have little effect on the axial resolution and excitation efficiency of the system and only low-order components require correction. The random aberrations varied in strength with the depth of the specimens, so the number of corrected Zernike modes is variable. A general formula is generated for the estimation of the number of modes that should be detected and corrected under different aberrations and different numerical aperture of the objective. The results can provide important guidance in the design and evaluation of AO units for TP-LSM systems.
关键词： Two-photon Light-sheet Microscopy,Aberration correction,Adaptive optics
更新于2025-09-23 15:21:01
-
An azimuthally-modified linear phase grating: Generation of varied radial carpet beams over different diffraction orders with controlled intensity sharing among the generated beams
摘要： Diffraction gratings are important optical components and are used in many areas of optics such as in spectroscopy. A diffraction grating is a periodic structure that splits and diffracts the impinging light beam into several beams travelling in different directions. The diffracted beams from a grating are commonly called diffraction orders. The directions of the diffraction orders depend on the grating period and the wavelength of the impinging light beam so that a grating can be used as a dispersive element. In the diffraction of a plane wave from a conventional grating, the intensities of diffracted beams decrease with increasing order of diffraction. Here, we introduce a new type of grating where in the diffraction of a plane wave, the intensity of a given higher order diffracted beam can be higher than the intensity of the lower orders. We construct these gratings by adding an azimuthal periodic dependency to the argument of the transmission function of a linear phase grating that has a sinusoidal profile and we call them azimuthally-modified linear phase gratings (AMLPGs). In this work, in addition to introducing AMLPGs, we present the generation of varied radial carpet beams over different diffraction orders of an AMLPG with controlled intensity sharing among the generated beams. A radial carpet beam is generated in the diffraction of a plane wave from a radial phase grating. We show that for a given value of the phase amplitude over the host linear phase grating, one of the diffraction orders is predominant and by increasing the value of the phase amplitude, the intensity sharing changes in favor of the higher orders. The theory of the work and experimental results are presented. In comparison with the diffraction of a plane wave from radial phase gratings, the use of AMLPGs provides high contrast diffraction patterns and presents varied radial carpet beams over the different diffraction orders of the host linear phase grating. The resulting patterns over different diffraction orders are specified and their differences are determined. The diffraction grating introduced with controlled intensity sharing among different diffraction orders might find wide applications in many areas of optics such as optical switches. We show that AMLPG-based radial carpet beams can be engineered in which they acquire sheet-like spokes. This feature nominates them for potential applications in light sheet microscopy. In addition, a detailed analysis of the multiplication of the diffraction pattern of an AMLPG by the 2D structure of a spatial light modulator is presented. The presented theory is confirmed by respective experiments.
关键词： radial carpet beams,diffraction gratings,optical switches,light sheet microscopy,azimuthally-modified linear phase gratings
更新于2025-09-11 14:15:04
-
[IEEE 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) - Honolulu, HI, USA (2018.7.18-2018.7.21)] 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) - Tracking Gene Expression via Light Sheet Microscopy and Computer Vision in Living Organisms
摘要： Automated tracking of spatiotemporal gene expression using in vivo microscopy images have given great insight into understanding developmental processes in multicellular organisms. Many existing analysis tools rely on the fluorescent tagging of cell wall or cell nuclei localized proteins to assess position, orientation, and overall shape of an organism; information necessary for determining locations of gene expression activity. Particularly in plants, organism lines that have fluorescent tags can take months to develop, which can be time consuming and costly. We propose an automated solution for analyzing spatial characteristics of gene expression without the necessity of fluorescent tagged cell walls or cell nuclei. Our solution indicates, segments, and tracks gene expression using a fluorescent imaging channel of a light sheet microscope while determining gene expression location within an organism from a Brightfield (non-fluorescent) imaging channel. We use the images obtained from the Arabidopsis thaliana root as a proof of concept for our solution by studying the effects of heat shock stress on CYCLIN B1 protein production.
关键词： computer vision,CYCLIN B1,light sheet microscopy,gene expression,Arabidopsis thaliana
更新于2025-09-09 09:28:46
-
Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy
摘要： Background: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. New method: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. Results: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. Comparison with existing methods: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. Conclusions: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.
关键词： Traumatic brain injury,Dystrophy,Structured illumination microscopy,Human induced pluripotent stem cell derived neurons,Lattice light sheet microscopy
更新于2025-09-04 15:30:14
-
Cancellation of Bessel beam side lobes for high-contrast light sheet microscopy
摘要： An ideal illumination for light sheet fluorescence microscopy entails both a localized and a propagation invariant optical field. Bessel beams and Airy beams satisfy these conditions, but their non-diffracting feature comes at the cost of the presence of high-energy side lobes that notably degrade the imaging contrast and induce photobleaching. Here, we demonstrate the use of a light droplet illumination whose side lobes are suppressed by interfering Bessel beams of specific k-vectors. Our droplet illumination readily achieves more than 50% extinction of the light distributed across the Bessel side lobes, providing a more efficient energy localization without loss in transverse resolution. In a standard light sheet fluorescence microscope, we demonstrate a two-fold contrast enhancement imaging micron-scale fluorescent beads. Results pave the way to new opportunities for rapid and deep in vivo observations of large-scale biological systems.
关键词： light sheet microscopy,Bessel beams,fluorescence imaging,droplet illumination,Airy beams
更新于2025-09-04 15:30:14