CaMPARI

CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) is a photoconvertible protein construct that enables imaging of the integrated calcium activity of large populations of cells over defined time windows.

Calcium is an important regulator of many cellular signaling events, and calcium indicators have found widespread use for imaging and measuring changes in Ca2+ levels associated with neural activity.  Typical genetically encoded calcium indicators (GECI) exhibit rapid response to Ca2+ concentration changes, which provides real-time information but limits studies to areas within a microscope’s field of view. Complementary methods to monitor larger cell populations and track calcium activity over defined time windows would open new research capabilities.

Inventors at Howard Hughes Medical Institute’s Janelia Research Campus have developed a new photoconvertible protein that enables imaging of the calcium activity history of large areas and populations of cells.  This new calcium integrator is based on EosFP, a fluorescent protein whose emission changes from green to red upon irradiation with UV-light (~400 nm).  By engineering libraries of EosFP variants, inventors have developed a protein that undergoes significantly faster green-to-red photoconversion (PC) only when calcium is present while PC light is applied. This permanent conversion records the calcium activity for all areas illuminated by PC-light.  Red fluorescence intensity correlates with calcium activity, thus integrating total levels during the PC-defined time window.  Optimized variants have been validated in cultured rat hippocampal neurons, with observable PC for 10 action potential trains and 2 s of PC exposure.  Further improvements should yield variants with improved contrast ratios, brightness and response kinetics.  CaMPARI complements existing calcium indicators by enabling measurements of the total calcium activity over large areas of cells and tissues.

Advantages:

• Image total calcium activity during defined time windows (gated by photoconversion light)
• Not restricted to the field of view of a microscope, as during real-time calcium imaging with e.g. GCaMP
• Enables higher-throughput calcium assays with cultured cells

Applications:

• Calcium activity imaging of across large cell popu-lations and/or tissues
• Labeling of “active” cells within a tissue (such as brain) during stimulus or behavior in model organisms
• Tracing of neurons based on their calcium activity level
• Integration of subcellularly localized calcium ac-tivity when targeted to specific subcellular locations

Publication:

Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. 
Fosque BF, Sun Y, Dana H, Yang CT, Ohyama T, Tadross MR, Patel R, Zlatic M, Kim DS, Ahrens MB, Jayaraman V, Looger LL, Schreiter ER Science. 2015 Feb 13;347(6223):755-60. doi: 10.1126/science.1260922. 

Patent Status:

Issued U.S. Patents 9,518,996 and 10,067,148

Opportunity:

Variants and packages of CaMPARI are available for purchase at Vigene Biosciences, available for non-profit research from Addgene, Penn Vector, and available for Commercial License. More recent variants may be available from Janelia. 

For inquiries, please reference:

Janelia 2013-016

Plasmids available from Addgene.org: