Optogenetics began with methods to alter neuronal activity with light, using e.g. channelrhodopsins. In a broader sense, optogenetic approaches also include the use of genetically encoded biosensors to monitor the activity of neurons or other cell types by measuring fluorescence or bioluminescence. Genetically encoded calcium indicators (GECIs) are used frequently to monitor neuronal activity, but other cellular parameters such as membrane voltage or second messenger activity can also be recorded optically. The use of optogenetic sensors is not restricted to neuroscience, but plays increasingly important roles in immunology, cardiology and cancer research.
The first experiments to measure intracellular calcium levels via protein expression were based on aequorin, a bioluminescent protein from the jellyfish Aequorea. To produce light, however, this enzyme needs the 'fuel' compound coelenteracine, which has to be added to the preparation. This is not practical in intact animals, and in addition, the temporal resolution of bioluminescence imaging is relatively poor (seconds-minutes). The first genetically encoded fluorescent calcium indicator (GECI) to be used to image activity in an animal was cameleon, designed by Atsushi Miyawaki, Roger Tsien and coworkers in 1997.[1] Cameleon was first used successfully in an animal by Rex Kerr, William Schafer and coworkers to record from neurons and muscle cells of the nematodeC. elegans.[2] Cameleon was subsequently used to record neural activity in flies[3] and zebrafish.[4] In mammals, the first GECI to be used in vivo was GCaMP,[5] first developed by Junichi Nakai and coworkers in 2001.[6] GCaMP has undergone numerous improvements, notably by a team of scientists at the Janelia Farm Research Campus (GENIE project, HHMI), and GCaMP6[7] in particular has become widely used in neuroscience. Very recently, G protein-coupled receptors have been harnessed to generate a series of highly specific indicators for various neurotransmitters.[8][9]
Design principles
Genetically encoded sensors are fusion proteins, consisting of a ligand binding domain (sensor) and a fluorescent protein, attached by a short linker (flexible peptide). When the sensor domain binds the correct ligand, it changes conformation. This movement is transferred to the fluorescent protein and the resulting deformation leads to a change in fluorescence. The efficiency of this process depends critically on the length of the linker region, which has to be optimized in a labor-intensive process. The fluorescent protein is often circularly permuted, i.e. new C-terminal and N-terminal ends were created. Single-wavelength sensors are easy to use for qualitative measurements, but difficult to calibrate for quantitative measurements of ligand concentration.
A second class of sensors relies on Förster resonance energy transfer (FRET) between two fluorescent proteins (FP) of different color. The shorter wavelength FP (donor) is excited with blue light from a laser or LED. If the second FP (acceptor) is very close, the energy is transferred to the acceptor, resulting in yellow or red fluorescence. When the acceptor FP moves further away, the donor emits green fluorescence. The sensor domain is typically spliced between the two FPs, resulting in a hinge-type movement upon ligand binding that changes the distance between donor and acceptor. The imaging procedure is more complex for FRET sensors, but the fluorescence ratio can be calibrated to measure the absolute concentration of a ligand. Read-out via fluorescence lifetime imaging (FLIM) of donor fluorescence is also possible, as the FRET process speeds up the fluorescence decay.
Advantages of optogenetic sensors
can be targeted to specific classes of cells (e.g. astrocytes or pyramidal cells). This allows for optical read-out without spatial resolution, e.g. fiber photometry from deep brain areas.[10]
can be targeted to sub-cellular compartments (e.g. synapses, organelles, nucleus) by fusing the indicator protein with specific anchoring domains, retention signals or intrabodies.
most indicators are green fluorescent, making it difficult to measure several cellular parameters simultaneously (multiplexing).
Classes of genetically encoded indicators
The calcium indicator GCaMP in its calcium-bound (top) and calcium-free form (bottom). When Ca-calmodulin (cyan) binds to M13, the conformation changes and the cpGFP barrel closes, enabling green fluorescence.
Indicators have been designed to measure ion concentrations, membrane potential, neurotransmitters, and various intracellular signaling molecules. The following list provides only examples for each class; many more have been published.
Intracellular signaling
Genetically encoded calcium indicators (GECI): A large class of tools, based on natural calcium binding proteins (calmodulin, troponin). Different affinities, kinetics, and colors (green, red) available. Read-out via fluorescence intensity (single wavelength indicators), FRET or BRET. Have been targeted to various organelles. Current version: JGCaMP8[13]
Fluorescent Biosensor Database, a fairly complete searchable list of published sensors and their basic properties, maintained by Jin Zhang's lab at UCSD.[34]
↑ Nakai J, Ohkura M, Imoto K (February 2001). "A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein". Nature Biotechnology. 19 (2): 137–141. doi:10.1038/84397. PMID11175727. S2CID30254550.
↑ Han J, Burgess K (May 2010). "Fluorescent indicators for intracellular pH". Chemical Reviews. 110 (5): 2709–2728. doi:10.1021/cr900249z. PMID19831417.
↑ Marvin JS, Shimoda Y, Magloire V, Leite M, Kawashima T, Jensen TP, etal. (August 2019). "A genetically encoded fluorescent sensor for in vivo imaging of GABA". Nature Methods. 16 (8): 763–770. doi:10.1038/s41592-019-0471-2. PMID31308547. S2CID196812412.
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