Fiber photometry has become a very popular method for in vivo calcium imaging due to its simple data output and system design.
You are probably asking, what components are needed to perform fiber photometry?
In short, you need two main categories of components to perform a successful fiber photometry experiment: biology and equipment.
Brains don’t naturally express genetically encoded calcium indicators (GECIs), meaning there are biological steps to perform fiber photometry in freely-behaving animals. First, you must express the genetic indicator in the brain; and second, you need to implant an optical cannula to collect fluorescent signals from the brain.
The first important step is to achieve optimal GECI expression in your animal model.
Mice are the most common animal model used for in vivo calcium imaging due to the advancement of genetic mice models (Daigle et al. 2018).
Neuroscientists employ two methods to express GECIs in the brain: viral expression and transgenic mouse models.
Viral expression involves injecting a virus encoding a GECI in the brain. This virus is linked to a gene of interest to target expression in a specific cell-type.
A crucial step associated with viral expression is testing varying dilutions of the virus to obtain optimal expression in the brain (Resendez et al. 2016). Too little expression can lead to no signal, and over-expression can lead to high background fluorescence (viz. noise) – or even cell death!
Neuroscientists employ viral expression to regulate GECI expression. This is useful because expression can vary depending on the brain region, cell-type, or virus. In addition, neuroscientists can use viral expression to express GECIs in brain projections to map neural circuits across brain regions.
In comparison to manual viral injections, transgenic mice models are designed to express the GECI throughout the entire brain (Dana et al. 2014). Depending on the transgenic model, GECI expression can vary from region to region, such that one region may express the GECI more than the other.
After successful GECI expression, you need to access the fluorescent signal inside the brain. But how can you see into the brain when it’s covered by both skin and skull?
Generally, this involves surgically implanting an optical cannula into the brain where the GECI is expressed.
Optical cannulas, which are used in fiber photometry experiments, enable light to be collected from the brain and delivered to a photodetector. Due to their simple design and lack of spatial resolution, optical cannulas are only capable of collecting one lumped signal or a population signal—providing little or no spatial resolution to image individual cells. Depending on the length of the optical cannula, they can be used to collect signal in shallow or deep brain regions. The surgery for implantation of optical cannulas is the least invasive surgery because of the compact design, which minimizes tissue damage. It is important to select low auto fluorescence cannulas, as this can affect your fiber photometry signal by adding background signal.
Now that you have all biological components setup, including optimal GECI expression and cannula implantation, you need equipment to deliver, extract, and record fluorescent signals from the brain of a freely-behaving animal.
A typical fiber photometry system is comprised of three main components:
- An optical fiber
- A light source and filter set
- A photodetector or an imaging device
A fluorescent signal is emitted from the GECI, and this is transmitted through the optical cannula. But how can the signal be collected?
First, you need a coupling between the optical cannula, light source, and imaging device. This coupling enables illumination of the GECI in the brain through the optical cannula and transmission of the emission signal to the camera. Coupling is achieved using an optical fiber. Similar to a cannula, it is important to select a low autofluorescence fiber to reduce any background signal that may contribute to the fiber photometry signal.
Light Source and Filter Set
Coupling enables you to deliver stimulation/light to and collect fluorescent signals from the brain. GECIs function by generating fluorescence signals, such that they have an excitation and emission spectrum (Grienberger & Konnerth, 2012). To excite GECIs and collect the emitting signal, you require two components: a light source and dichroics/filters.
For excitation of the GECI, LED light sources are commonly selected for fiber photometry since only relatively modest optical power is required. Of course, there is a balance between too little and too much power: either not getting enough signal or photobleaching your sample.
Importantly, the correct excitation wavelength must be selected. For example, GCaMP excitation is blue (~470nm) and emission is green (~530nm). And this is where the second component is necessary. Dichroics and filters allow proper transmission of the correct excitation wavelength to the brain and transmission of the correct emission signal from the brain to the imaging device.
For fiber photometry experiments, individuals may use an isosbestic wavelength, such as 405nm, to control for any movements or artifacts that may contribute to the fiber photometry signal (Kim et al. 2016).
Photodetector or Imaging Device
Lastly, you need to detect, record, and analyze fluorescent signals from the brain. This is made possible using a photodetector or an imaging device. Three types of devices are used for fiber photometry systems: 1) scientific camera, 2) PMT, and 3) photodetector. In the standard setup for single region imaging, individuals will use a photodetector or PMT with a lock-in amplifier for fast acquisition and low signal-to-noise; however, this method is limited to investigating one individual signal. If you want to perform fiber photometry in multiple brain areas, you will have to use multiple sets of photodetectors or PMT’s (which are expensive), or you may be better off using a scientific camera to measure multiple fiber photometry signals simultaneously.
Successful fiber photometry experiments are a balancing act between the biology and equipment. Luckily, both the biology and equipment are constantly being optimized for better performance and ease of use.
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