IVIS Methods and Protocols

Basic Imaging Tips

1)  Biological science: Preparing your experiment

  1. Hash out your experiment with the Cadre Director to make up one's mind optimal combination of animals and imaging products.
  2. Plate a black well plate (plate purchasing information) with dilutions of your imaging product in vitro to ensure your point is detectable earlier attempting in vivo experiments.
  3. Recommended animals: mice are preferred as we currently carry equipment for these animals; if using rats, researchers must bring their own nose cones if they wish to use the XGI-eight Anesthesia Organization.
  4. In our IVIS Core, we have not yet used larger mammals, such as rabbits, cats, or chinchillas, but shallow tissue in these animals (up to ~ii.5 cm maximum depth for the strongest signal) has been imaged successfully in other IVIS Spectrum Instruments. Feel free to contact the Core Director with other animal questions.

Some Suggested Protocols:

  • Run into ASC website for injections techniques
  • D-luciferin Preparation Protocol
  • Caliper Intraperitoneal-Injections

two)  Acquisition: Optimizing and Imaging Protocol

Bioluminescence

Note: if you are performing an experiment using both bioluminescent and fluorescent reporters, e'er paradigm fluorescence Before bioluminescence equally brilliance can emit in the same range every bit some fluorophores (particularly reds) and may interfere with fluorescent signal.

    1. Determine your optimal in vivo imaging time by taking incremental images. Typically for firefly luciferase, luminescence does not accomplish a detectable radiance until afterwards x minutes .
    2. Use the command panel to either manually set or use the software'due south Imaging Wizard (under sequence setup) to automatically set imaging parameters. Note that luminescent images often crave longer exposure times and a lower Fstop (a wider discontinuity allows more light to attain the CCD) than fluorescent images. Ideally, images should have no longer than 5 minutes of exposure time. Auto exposure is recommended to observe optimal exposure conditions as set up automatically by the CCD photographic camera.
Affect sensitivity
Image from Caliper
  1. Use Sequence Setup to take multiple images while changing i parameter per paradigm.
    Cull the fourth dimension point at which the measured signal value in full photon flux "plateaus" (remains nearly the same value) every bit the post-obit image.

Fluorescence

  1. Make up one's mind maximum signal strength either manually or automatically using the Imaging Magician.  Note that fluorescent images often require a quicker exposure time, a larger Fstop, and specific emission and excitation filters.  Also keep in mind that animate being tissue can generate autofluorescence, and so background subtraction may be necessary
  2. Have a sequence of images (if the wizard has not already prompted you to practice then)
  3. Cull best filter set image (by comparing indicate to dissonance ratios of all images in sequence) for analysis or move on to groundwork subtraction features:

– Adaptive FL subtraction
– Image Math Tool
– Spectral Unmixing

Advanced Imaging Tips

3D Reconstruction

  1. Using Living Image 3.2 software'southward 3D reconstruction feature, cell number can be quantified. To do this, an in vitro measurement of a stably integrated prison cell line of interest must be taken using the camera. Because of algorithms in the software that correct for tissue obstruction, this in vitro measurement can and then be correlated to the betoken measurement obtained from a 3D reconstruction.
  2. The Living Paradigm 3.two software has the ability to locate the "center of mass" of signal quite specifically relative to the animate being topography that is generated for a 3D reconstruction. Typically, at the end of an experiment, it is of import to sac and dissect the fauna to ostend betoken location. The software provides millimeter measurements in the side panels, which tin be used to decide the depth of betoken from diverse surfaces of the animal.
  3. Organs can exist imaged separately after dissection for comparison with in vivo images and for confirmation of source location.
  4. Call up that animals may be euthanized in the IVIS Core Facility (W914), only dissections must take place elsewhere.

How to Schedule

Please login to iLab system to schedule equipment fourth dimension or services. For new users delight follow the steps outlined in Information for New Users.

Contact

Francesca Seta, PhD
Cadre Director
Evans Biomedical Research Center, X-Edifice
650 Albany St. seventh Floor, Room X-720
Boston, MA 02118
(617) 358-7814| setaf@bu.edu

Location

IVIS Imaging Core
Boston University Medical Campus
Center for Advanced Biomedical Research, W-Building
700 Albany St., 9th Flooring
Boston, MA 02118

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