Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK
A procedure for cell type-specific expression and imaging of a FRET-based ATP biosensor in neurons and astrocytes within mouse hippocampus and cortex tissue slice cultures is described. Imaging is accomplished using an upright epifluorescence microscope with emission splitter for simultaneously capturing the separate donor and acceptor emission channels.
Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores
Sub-diffraction limited imaging of Bacillus subtilis spores is demonstrated using super-resolution Structured Illumination Microscopy (SIM), allowing for characterization of germinosomes (clusters of germination proteins) and spore membranes with spatial resolution greater than that provided by traditional widefield epifluorescence imaging.
Imaging the Human Immunological Synapse
Time-lapse widefield epifluorescence imaging with subsequent deconvolution analysis is applied towards visualizing immunological synapse (IS) formation and trafficking activity in a standard cell synapse model composed of Jurkat T lymphocyte cells and Raji lymphoblast-like cells presenting the superantigen Staphylococcus enterotoxin E.
Conducting Multiple Imaging Modes with One Fluorescence Microscope
A multimodal microscope configuration for widefield epifluorescence, super-resolution single molecule localization microscopy (SMLM), and single molecule Förster Resonance Energy Transfer (smFRET) based on a commercial system is described. Additionally, both SMLM and smFRET can be performed in a TIRF configuration for improved signal-to-noise imaging and optical sectioning.
Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data
To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.
Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread
Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Methodologies are presented that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.
Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia
Changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. Intracellular calcium is imaged using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.
In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis
The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers.
Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy
This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.
Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging
A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.
- BioStation IM Time Lapse Imaging System
Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.
Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants
Approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.
An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
A protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time.