Real-time monitoring of brain activity has become the gold standard for behavioral and circuit neuroscience. The ability to link signaling in vivo to a subject’s behavior provides powerful insight into how the brain functions. Fiber photometry is a highly versatile imaging technique that allows researchers to monitor the activity of genetically-defined cell populations or the release of specific neurotransmitters in vivo.
Put simply, fiber photometry (FP) measures light emitted by specialized fluorescent biosensors as a proxy for neuronal activity. In a typical FP experiment, a viral vector is injected into a specific region of the brain to drive the expression of a genetically-encoded biosensor. An optical fiber is then chronically implanted into the same region so that fluorescence can be measured in vivo. A key advantage of this approach is that it is possible to image in deep brain areas. Further, multiple optical fibers can be implanted into the same subject to monitor activity bilaterally or in interconnected brain regions. Optical Fibers are then attached to an imaging system using a lightweight cord that allows for recording while subjects are freely-behaving.
Fiber photometry can be used to record a wide variety of biological signals depending on the biosensor used and how it is expressed. Genetically-encoded fluorescent biosensors used for FP have two key properties. First, that they fluoresce upon excitation by a specific wavelength of light, and second that their ability to fluoresce is highly dependent upon binding to a specific ligand. This unique property allows scientists to interpret an increase in fluorescent emission as an increased concentration of a specific molecule. Biosensors can be expressed constitutively to measure the activity in all cells within a region of interest, or in a cell-type specific manner using transgenic animal lines or targeted promoters.
There are two main classes of biosensors that are compatible with fiber photometry; genetically-encoded calcium indicators (GECIs), and neurotransmitter sensors. GECIs, the most common of which is GCaMP, are expressed in the cytosol and measure changes in intracellular Ca2+ levels. Intracellular Ca2+ dynamics are interpreted as a proxy for neuronal firing or depolarization and can be used to understand the activity of specific cell populations.
A newer generation of sensors that bind to specific neurotransmitters and signaling molecules are now widely used with fiber photometry. Typically expressed on cell membranes, neurotransmitter biosensors measure the extracellular concentration of target molecules to provide insight into how and when specific neurotransmitters are released.
While most biosensors are green fluorescent protein-based, more and more red fluorescent protein-based sensors are being developed, allowing for simultaneous recording of multiple sensors. Using a combination of sensors makes it possible to record from multiple populations at the same time, pre- and post-synaptic responses, etc., expanding the potential applications for fiber photometry.
Fiber photometry measures bulk changes in fluorescent intensity to provide information on the collective firing patterns of neuronal populations in real-time. Akin to local field potential, signals can be interpreted as the average activity of indicator-expressing cells, or release of a neurotransmitter. Changes in activity can be measured over the course of milliseconds, minutes, days, and even weeks.
Photometry is often coupled with behavioral assays and other readouts to understand the link between brain activity and output. By recording simultaneously, researchers can correlate signals in the brain to certain behaviors or responses to stimuli, and assess how these signals change over time. FP can also be used in combination with optogenetics, electrophysiology, chemogenetics, and pharmacology to assess how circuit manipulations alter neuronal activity.
| 2-photon | 1-photon | Fiber Photometry | |
|---|---|---|---|
| Cell-type specific recording | x | x | x |
| Deep brain recordings | x | x | x |
| Single cell resolution | x | x | |
| Multi-region recordings | x | ||
| Pre/Post-synaptic imaging | x | ||
| Integrated optogenetics | x | x | x |
| Freely-moving subjects | x | x | |
| Long Term (multi-week) recording | x | ||
| Data processing complexity | *** | ** | * |
| System and setup cost | $$$ | $$ | $ |
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