What does GCaMP detect?

What does GCaMP detect?

The GCaMP6 family of GECIs is a collection of ultrasensitive, green fluorescent indicator proteins that enable reliable detection of single action potential responses in vivo and facilitate the measurement of synaptic calcium signals.

What are calcium indicators?

Chemical indicators are small molecules that can chelate calcium ions. All these molecules are based on an EGTA homologue called BAPTA, with high selectivity for calcium (Ca2+) ions versus magnesium (Mg2+) ions. This group of indicators includes fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1.

How does GCaMP detect calcium?

When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using virally transfected or transgenic cell and animal lines.

How does Fura-2 work?

Fura-2, an aminopolycarboxylic acid, is a ratiometric fluorescent dye which binds to free intracellular calcium. The use of the ratio automatically cancels out confounding variables, such as variable dye concentration and cell thickness, making Fura-2 one of the most appreciated tools to quantify calcium levels.

What is in vivo fiber photometry?

Fiber photometry is an in vivo calcium imaging method that detects average fluorescence intensity changes. Thus, this method is used to measure population neural activity in a freely-behaving animal (Cui et al. 2014).

What is GCaMP6m?

Calcium indicator GCaMP6m lentivirus is a preparation of highly purified lentiviral particles ready-to-use for monitoring calcium signal in virtually any type of mammalian cells. GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology.

How is intracellular calcium detected?

Calcium detection and measurement in living cells can be achieved using a variety of techniques. Three of these are based on the optical detection of changes in luminescence, fluorescence or absorbance of an organic indicator produced on binding with calcium ions.

What are GECIs?

GECIs measure calcium, the key signal molecule underlying cell functions such as heart, vessel, and airway contraction, lung secretion, autonomic neurotransmission, and immunocyte function. GCaMP, originally developed by Junichi Nakai is an example of a GECI.

Is GCaMP ratiometric?

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins.

How do you take a picture in Fura 2?

Imaging Protocol Place a drop of oil on the objective, place the chamber on the microscope stage and focus on the cells using transmitted light. Examine the fluorescence of the cells using illumination at 340 and at 380 nm using the eyepieces. Resting cells should be dim at 340 and bright at 380.

What is ratiometric imaging?

Ratiometric fluorescence is the method where intensities at two or more wavelengths of an excitation or emission spectrum are measured to detect changes to local environment. Typically, a probe is used that is specifically sensitive to an environmental parameter such as ion concentration, pH, viscosity, or polarity.

Is fiber a photometry imaging?

Fiber photometry is an imaging method that enables scientists to image population-level neural activity in the brain of freely-behaving animals.

What is the difference between calcium sensors and FRET sensors?

As opposed to single-wavelength sensors, FRET -based calcium sensors are based on two fluorescent proteins linked by a peptide and a calcium binding domain (e.g., CaM or Troponin). Calcium binding brings the FPs closer together, increasing FRET efficiency between the two proteins.

What are the different types of calcium indicators?

Genetically encoded calcium indicators (GECIs) can be divided into three different classes ( Table 1) ( 49 ), two of them have just been described. Class 3 indicators (“chameleon”-type sensors) contain two fluorescent proteins and are applied to measurements involving fluorescent resonance energy transfer (FRET).

What is the role of GFP in fluorescent calcium indicators?

GFP is used as scaffolding for the most recent fluorescent calcium indicators, namely genetically encoded calcium indicators (GECIs) like GCaMP ( 26 ). In those proteins, circularly permuted forms of GFP are fused to calmodulin (CaM) and the M13 domain of the myosin light chain kinase (MLCK).

Can GCaMP indicators be used to indicate calcium levels?

Thus, GCaMP indicators can be used to indicate calcium levels in real time. GCaMP variants have a range of on/off kinetics, and can be used to measure different types of cell activity. Specifically, the fastest variant (GCaMP6f) has a 2.6-fold higher Ca2+ affinity (lower kd) than the slow variant (GCaMP6s) (Table 1).