Imaging Tips
Basic Imaging Tips
Recommended Protocol:
- Discuss your experiment with the Core Manager or Directors to determine optimal combination of animals and imaging products.
- Plate a black 96-well plate with dilutions of your imaging product in vitro to ensure your signal is detectable before attempting in vivo studies.
Recommended animals:
-mice are preferred as currently we only carry equipment for these animals
-rats are currently being used successfully, but researchers must bring their own nose cones
-At BU, we have not yet used larger mammals, such as rabbits, cats, or chinchillas, but shallow tissue in these animals (up to ~2.5 cm maximum depth for the strongest signal) has been imaged successfully in other IVIS Spectrum Instruments. Feel free to contact the Core Manager with other animal questions.
Recommendations for two-dimensional Imaging
Bioluminescence
*Note: if you are performing an experiment using both bioluminescent and fluorescent reporters, always image fluorescence BEFORE bioluminescence as luminescence can emit in the same range as some fluorophores (particularly reds) and may interfere with fluorescent signal
1) Determine your optimal in vivo imaging time by taking incremental images
- typically, luminescence does not reach a detectable radiance until after 10 minutes
Image from G. Murphy
2) Use the control panel to either manually set or use the software’s Imaging Wizard (under sequence setup) to automatically set imaging parameters
-Note that luminescent images often require longer exposure times and a lower Fstop (a wider aperture allows more light to reach the CCD) than fluorescent images
- ideally, images should take no longer than 5 minutes of exposure time
Image from Caliper
3) Use Sequence Setup to take multiple images while changing one parameter per image
4) Choose the best image of the sequence and note parameters
Fluorescence
1) Determine maximum signal strength either manually or automatically using the Imaging Wizard
-Note that fluorescent images often require a quicker exposure time, a larger Fstop, and specific emission and excitation filters
-Also keep in mind that animal tissue can generate a great deal of autofluorescence, so background subtraction may be necessary
2) Take a sequence of images (if the wizard has not already prompted you to do so)
Image from S. Narasimhan
3) Choose best image for analysis or move on to background subtraction features:
- Adaptive FL subtraction
- Image Math Tool
- Spectral Unmixing
Advanced Imaging Tips
DLIT Bioluminescent three-dimensional Reconstruction
*Voxels within 1% of the max intensity are displayed
Suggested Imaging Tools/Reagents
Caliper Website and Pdfs: http://www.caliperls.com/products/reagents/in-vivo-imaging-reagents/
- Near Infrared Fluorescent Reagent for in vivo imaging: xenolight-dir-v2
- Xenolight Fluorescent Dye Kits for in vivo imaging: xenolight-dir-v21
Boston University CReM Vector Core (and IVIS Core subset): http://www.bumc.bu.edu/stemcells/crm-core-facilities/vector-core/
- the IVIS Imaging Core provides free aliquots of bioluminescent and fluorescent plasmids as a subset of the Vector Core and intends to eventually offer virus at a designated price.
