ABSTRACT
Indicators for Ca2þ ...................................................................................... 101 4.2 Ca2þ Indicators Using Single GFP Variants............................................... 102 4.3 Ca2þ Indicators Using Circularly Permuted GFP Variants ........................ 102
4.3.1 G-CaMP ........................................................................................... 102
4.3.2 Pericam............................................................................................. 103
4.4 Ca2þ Indicators Utilizing Pairs of GFP Variants That Permit FRET ....................................................................................... 105
4.4.1 Procedure for Dual Emission Ratio
Imaging of YCs................................................................................ 107
4.5 Monitoring Rodent Neural Activity with
GFP-Based Fluorescent Ca2þ Indicators ...................................................... 108 References............................................................................................................. 109
Green fluorescent protein (GFP)-based fluorescent indicators for Ca2þ offer significant promise for monitoring Ca2þ in previously unexplored organisms, tissues, organelles, and submicroscopic environments because they are genetically encoded,
function without cofactors, can be targeted to any intracellular location, and are
bright enough for single-cell imaging [1-3]. These indicators may be either single
GFP variants [4], circularly permuted GFP variants [5, 6], or pairs of GFP variants
that permit fluorescence resonance energy transfer (FRET) [7, 8]. This review
describes the design and construction of these three types of Ca2þ indicators, as well as their potential uses and limitations. It also focuses on recent reports on
improvements and applications of Ca2þ indicators that have appeared in the last few years. These reports demonstrate progress toward a real understanding of the
dynamic aspects of Ca2þ homeostasis.
