Last Updated on April 5, 2021
Fiber Photometry is a rapidly advancing field, with biosensors for more analytes and with better sensitivity being announced almost every month. We would like to share information about sensors that should be compatible with fiber photometry when using excitation with blue (~480 nm) light and measuring green (~525 nm) fluorescence. This is the most commonly used wavelength pair and is offered with TeleFipho wireless fiber photometry.
We will update this guide as more information becomes available.
Overview of Fluorescent indicators: Structure and considerations for use.
Genetically encoded fluorescent indicators (GEFIs) are used in conjunction with fiber photometry to report on changes in concentrations of molecules in vivo in real-time.
Most fluorescent biosensors comprise a fluorescent protein yoked to an analyte binding protein, constructed so that binding of the analyte causes a dramatic increase in fluorescence.
Akerboom, Rivera, Guilbe, Malavé, Hernandez, Tian, Hires, Marvin, Looger, Schreiter ER / CC BY (https://creativecommons.org/licenses/by/3.0)
When used with fiber photometry in behaving animals, the sensors are usually introduced by injecting viral expression vectors. The virus is used to both express the sensor and to control its location: targeting sequences allow the researcher to choose a specific cellular subtype, and even the cellular localization, such as axon or soma, where the sensor will be expressed. Anterograde and retrograde localization can also target only a specific projection or circuit in a target region. Transgenic animals are also available for expressing some GEFIs.
Binding kinetics helps determine the range of concentrations the sensor will respond to, and its ability to report fast events. A sensor with high affinity (low Kd) and a long dissociation time can measure very low concentrations of a molecule, but this happens at the expense of being able to resolve more frequent events and a narrower useful range of concentrations. Fast dissociation improves time resolution, but sensitivity usually suffers.
The brightness of the sensor, partially expressed as the ratio of the increase in fluorescence when bound to the analyte compared to baseline (∆F/F or ∆F/F0), is the other major factor to consider. Brighter sensors can generate a useful signal when expressed at lower levels or when used with less illumination when compared to less bright sensors. They can also be used with narrower fibers. Some of our users are stepping down to 250 microns from 400-micron core diameter when using the latest generations of GCaMP type sensors for fiber photometry.
Most biosensors are already available from AddGene: some as plasmids, others as aliquots of ready-to-use viral vectors. The newest biosensors listed here can be sourced directly from the laboratories which invented them. We included the best source we could find, and the original publication describing the sensor in the table below.
|Calcium Sensors||Affinity (Kd or EC50)||dissociation
Kinetics (Mean life,
|∆F/F0 (% increase)||Source for vector or plasmid||Reference|
|GCaMP6s||147 nM||1796 ms||1680|
|GCaMP6f||375 nM||400 ms||1314|
|jGCaMP7s||68 nM||1260 ms|
|jGCaMP7f||150 nM||270 ms||3100|
|jGCaMP8f||334 nM||27 ms||7880|
|jGCaMP8m||108 nM||55 ms||4570|
|jGCaMP8s||46 nM||272 ms||4950|
The GCaMP6 series of genetically encoded Ca2+ indicators (GECIs) are the most popular tools for examining action potentials and have been used extensively with TeleFipho photometry. GCaMP6f is optimized for fast decay kinetics, necessary for monitoring quick events, while GCaMP6s have higher sensitivity and slower decay kinetics. If you are starting a new project, consider the latest generation – jGCaMP7 – which offers higher sensitivity and a larger range of kinetics.
The jGCaMP7 series was introduced in 2019 as a collaborative effort between Loren Looger at Janelia and other research institutes. The jGCaMP7 GECIs have several-fold higher ∆F/F0 and a wider range of kinetics when compared to the earlier GCaMP6 sensors. Some GCaMP7 variants that will interest fiber photometry users include jGCaMP7s (highest sensitivity, but slower kinetics), and jGCaMP7f which has the fastest kinetics. We hope to have calcium data generated using jGCaMP7s and TeleFipho wireless fiber photometry soon.
The jGCAMP8 series from The Looger Lab and the GENIE Project Team at HHMI Janelia was introduced in late 2020 and is the most recent set of GECIs available with improved sensitivity and speed. Compared to jGCAMP7f, the new series all have a faster rise time. 8f (fast) has a 4x faster rise time and a 2.5x faster decay time. 8m (medium) again has about a 4x faster rise time but is also 3.5x more sensitive. 8s (sensitive) is 2x more sensitive and 2x faster. A bonus of using these newer, brighter sensors for fiber photometry is that narrower fibers can be used. Some of our users are stepping down from 400 microns to 250-micron core diameter fibers, allowing for less traumatic surgeries while targeting smaller areas.
GCaMP-X The calmodulin GCaMP based calcium sensors have been shown to cause side effects during some in-vivo uses, such as interference with the function of L-type calcium channels, nuclear accumulation, and cytotoxicity. Changes largely addressed these issues to the design of GCaMP-X.
Dopamine is rapidly becoming the second most common target for imaging and photometry in neuroscience thanks to two sensors introduced in 2018, dLight and GRABDA. The intensity of illumination used with dLight and GRABDA is typically 20 – 30 μW, the same range as is used with GCaMP6.
(Kd or EC50)
τ = 1/Koff)
|Source for vector or plasmid||Reference|
|dLight1.2||770 nM||90 ms||340|
|GRABDA1m||130 nM||700 ms||90|
|GRABDA1h||10 nm||2500 ms||90|
dLight1.1 and dLight1.2, developed by the Tian lab, have both been used extensively with fiber photometry, with settings similar to those used for GCaMP6.
GRABDA DA2M, DA2H
GRABDA (GPCR-Activation Based DA) was first introduced by Yulong Li’s lab in 2018 and has just been updated to increase Δf/f and increase the range of kinetics. The recent versions are DA2H (high affinity) and DA2M (medium affinity). Both GRABDA2m and GRABDA2H have already been used with fiber photometry, but so far results have only been communicated via preprints.
Norepinephrine and Serotonin
More from the Yulong Li lab, though as of yet their characterization is only available through preprints. GRABNE1m and GRAB5-HT1.0 have both already been used to measure norepinephrine and serotonin via fiber photometry in mice.
(Kd or EC50)
( τ = 1/Koff))
|GRABNE1h||Norepinephrine||83 nM||2000 ms||130|
|GRABNE1m||Norepinephrine||930 nM||750 ms||250|
|GRAB-5HT1.0||Serotonin||22 nM||3.1 s||280|
|iSeroSnFr||Serotonin||EC50 1.5 µm|
Biosensors for endo cannabinoids (GRABeCB), ATP, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), somatostatin (SST), vasopressin/oxytocin, ghrelin, and orexin were also announced by the Li lab at Neuroscience 2019, and are still being validated. The best way to keep up with the Li lab may check #GRABSensors on Twitter!
More information on iSeroSnFr from the Tian lab should be available soon.
There are two main sensor types available for monitoring glutamate: iGluSnFR and iGlu. The original iGluSnFR has slower kinetics, while iGluf (fast) and iGluu (ultrafast) are much faster. The new SF-iGluSnFR variants offer higher brightness and a range of different kinetics compared to the original.
iACHSnFR is one of the most recent GEFIs created by the Loren Looger lab at Janelia, along with collaborators.
GACH and GRABACh3.0
While the initial version of the GRAB type acetylcholine indicator (GACH) was not sensitive enough for measuring physiological levels of ACh using fiber photometry (personal communication), the version described in a preprint from Dec. 2019 (ACh3.0) has been used successfully with fiber photometry. The Li lab is also supplying researchers with an even newer version, ACh4.3.
Adenosine and ATP (Extracellular)
|Sensors||Analyte||Affinity (Kd or EC50||dissociation
(τ = 1/Koff)
|∆F/F0 (% increase)||Source for vector or plasmid||Reference|
|GRABATP1.0||ATP||EC50 ~45 nM||9 ms||500-1000|
|iATPSnFR1||ATP||EC50 of ~50 nM||190|
|GRABAdo||Adenosine||60 nM||63 ms||120|
Peng et al. monitored adenosine in the mouse basal forebrain using fiber photometry and GRABAdo, also called Ado1.0
Please let us know if you have any corrections or additions to this list!