Automatic Cell Segmentation with Zymomonas mobilis

Zymomonas Mobilis ZM4 is a well-known industrial gram-negative bacterium known for its high sugar uptake bioethanol production. With single-cell imaging and machine learning algorithms advancing rapidly, it will be much easier to understand the metabolic system of the bacterium. A pre-cursor to metabolic manipulation of the bacterium is to evaluate the cell growth of various populations by using an inducible CRISPR-interference (CRISPRi) toolset on fluorescent and metabolic targets to determine the construction of strains capable of manipulating cell metabolism to increase bioethanol production. To evaluate cell growth, we implemented single-cell time-lapse microscopy, which revealed population heterogeneity in knockdown rate. These observations led to using a cell segmentation program (CyAn) to analyze biomass and growth rates generated from metabolic manipulations. This program was coupled with the development of cell tracking software for Z. Mobilis using dilated Convolutional Neural Networks (dCNNs) to simplify the cell tracking pipeline and to open new avenues of tracking for the future.</p

Extended Lattice Light-Sheet with Incoherent Holography

Recently, lattice light-sheet instruments and imaging technology have shown a great improvement in exploring the dynamics of life at subcellular resolution. However, to reconstruct 3-dimensional structures the objective lens or the sample stage are invariably moved. Invention of incoherent holography lattice light-sheet (IHLLS) using FINCH provides quantitative information on the state and size of subcellular changes of neurons over the visible spectrum. The technique allows both faster three-dimensional amplitude as well as phase imaging without moving either sample stage or the detection objective. Amplitude and phase measurements provide intrinsic instrumental simplicity, larger scanning area, and higher resolution when compared to the original LLS schemes. IHLLS is built as an additional detection arm of an original lattice light-sheet (LLS) system. In this chapter, we review the evolution of IHLLS to address its feasibility and limitations

Digital holography applications in ophthalmology, biometry, and optical trapping characterization

This dissertation combines various holographic techniques with application on the two- and three-dimensional imaging of ophthalmic tissue, fingerprints, and microsphere samples with micrometer resolution. Digital interference holography (DIH) uses scanned wavelengths to synthesize short-coherence interference tomographic images. We used DIH for in vitro imaging of human optic nerve head and retina. Tomographic images were produced by superposition of holograms. Holograms were obtained with a signal-to-noise ratio of approximately 50 dB. Optic nerve head characteristics (shape, diameter, cup depth, and cup width) were quantified with a few micron resolution (4.06 -4.8 microns). Multiple layers were distinguishable in cross-sectional images of the macula. To our knowledge, this is the first report of DIH use to image human macular and optic nerve tissue. Holographic phase microscopy is used to produce images of thin film patterns left by latent fingerprints. Two or more holographic phase images with different wavelengths are combined for optical phase unwrapping of images of patent prints. We demonstrated digital interference holography images of a plastic print, and latent prints. These demonstrations point to significant contributions to biometry by using digital interference holography to identify and quantify Level 1 (pattern), Level 2 (minutia points), and Level 3 (pores and ridge contours). Quantitative studies of physical and biological processes and precise non-contact manipulation of nanometer/micrometer trapped objects can be effectuated with nanometer accuracy due to the development of optical tweezers. A three-dimensional gradient trap is produced at the focus position of a high NA microscope objective. Particles are trapped axially and laterally due to the gradient force. The particle is confined in a potential well and the trap acts as a harmonic spring. The elastic constant or the stiffness along any axis is determined from the particle displacements in time along each specific axis. Thus, we report the sensing of small particles using optical trapping in combination with the digital Gabor holography to calibrate the optical force and the position and of the copolymer microsphere in the x, y, z direction with nm precision

Deformation Measurements of Neuronal Excitability Using Incoherent Holography Lattice Light-Sheet Microscopy (IHLLS)

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective

Single calcium channel nanodomains drive presynaptic calcium entry at lamprey reticulospinal presynaptic terminals [preprint]

Synchronous neurotransmission is central to efficient information transfer in neural circuits, requiring precise coupling between action potentials, Ca2+ entry and neurotransmitter release. However, determinations of Ca2+ requirements for release, which may originate from entry through single voltage-gated Ca2+ channels, remain largely unexplored in simple active zone synapses common in the nervous system. Understanding these requirements is key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurological and neuropsychiatric disorders. Here, we present single channel analysis of evoked active zone Ca2+ entry, using cell-attached patch clamp and lattice light sheet microscopy over active zones at single central lamprey reticulospinal presynaptic terminals. Our findings show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising subtypes N-type (CaV2.2), P/Q-type (CaV2.1) and R-type (CaV2.3), available to gate neurotransmitter release. Significantly, of this pool only 1-6 (mean of 4) channels open upon depolarization. High temporal fidelity lattice light sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations between action potentials and stochastic variation of precise locations of Ca2+ entry within the active zone. Further, Ca2+ channel numbers at each active zone correlate to the number of presynaptic primed synaptic vesicles. Together, our findings indicate 1:1 association of Ca2+ channels with primed vesicles, suggesting Ca2+ entry via as few as one channel may trigger neurotransmitter release

Live Cell Light Sheet Imaging with Low- and High-Spatial-Coherence Detection Approaches Reveals Spatiotemporal Aspects of Neuronal Signaling

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca2+ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca2+ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca2+ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches