Overview of Multiphoton/Electrophysiology Core Facility Overview

Overview of the Multiphoton/Electrophysiology Core Facility

Our Mission

The goal of the Multiphoton Imaging/Electrophysiology Core is to provide instrumentation for analyzing protein localization, protein dynamics, and protein-protein interactions with high resolution. This facility will also allow users to perform time-lapse imaging of multiple fluorophores in living cells and tissues, and combine high resolution imaging of fluorescently tagged proteins or ion indicator dyes with electrophysiological monitoring of electrical activity.

Core Service

This core facility contains a multi-photon confocal microscope that allows high resolution imaging of labeled cell components in three dimensional space. The system consists of a Zeiss LSM 510 motorized upright microscope, a META scanning module, Coherent Chameleon tunable phased NIR laser for two-photon excitation, two non-descanned detectors for multi-photon work, and a motorized stage for time lapse imaging.

The LSM 510 can be configured with the META detector plus 2 traditional detectors for fluorescent or reflected light imaging. The META detector can also be used as a unique spectral detector as well as a conventional “band-pass” detector. All 3 detectors can be recorded and displayed simultaneously. This system employs PC-based user interface to control the microscope, laser excitation, scanning and image acquisition, and is capable of digitally capturing up to 12 bit 2K x 2K pixel images, while the LSM software controls image data acquisition and processing. The system can perform Fluorescent Recovery after Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET), and quantitative co-localization. The META detector provides multi-spectral imaging. The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of

in vivo imaging of fluorescent dyes or proteins. The facility also has the capability to couple high resolution imaging with electrophysiological recording of currents and voltages. Electrophysiological data can be acquired with an Axon Instruments Axopatch 200B patch amplifier, Axon Instrument DigiData 1440A A-D converter coupled to a stand alone PC. In addition, there is a Master 8 timer for applying controlled stimuli, a picrospritzer for locally applying drugs, and a video camera module for IR-DIC imaging. Three interchangeable temperature-controlled stages are available for use in live cell/tissue imaging, computer controlled multisite recording, or in vivo imaging.

In addition, the facility has a dedicated image analysis computer running Imaris software from Bitplane for performing deconvolution, 3D reconstruction, isosurface rendering, filament tracing/particle tracking, and quantitative co-localization. The Imaris suite of programs has been designed to accept images in many different formats, include Tiff series, BMP series, and images collected with Leica, Olympus, and Zeiss acquisition software. The available modules are Imaris suite, ImarisXT, Filament tracer, and AutoDeblur. By using ImarisXT, users can develop their own, task-oriented algorithms for Imaris, or use analysis modules developed by others. AutoDeblur is a deconvolution software that produces extremely high-quality results through image restoration.

Contact Information

Core investigators should contact Nadia Fakhari, MS (nfakhar1@jhmi.edu), the facility manager, or Dwight Bergles, Ph.D. (core director) (dbergles@jhmi.edu) to discuss their project and facility procedures; use of microscope must be scheduled in advance.

Click here to see the current schedule for the microscope.