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If someone were to describe in vivo calcium imaging to you, it might sound quite simple.

When you dig deeper, you start to appreciate the complexity associated with the biology and equipment to perform calcium imaging in freely-behaving animals.

You’re probably asking, what are the necessary components to perform in vivo calcium imaging?

Biological Components

Brains don’t naturally express genetically encoded calcium indicators (GECIs), meaning there are biological steps to perform calcium imaging in freely-behaving animals. First, you must express the genetic indicator in the brain; and second, you need to implant an imaging probe to collect fluorescent signals from the brain.

     Genetic Sensor Expression

The first and most important step is achieving 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 (1); however, calcium imaging has slowly progressed in rats and non-human primates (2,3).

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 (4). Too little expression can lead to no signal, and over-expression can lead to high background fluorescence – 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 (5). 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. Neuroscientists examining large cortical areas use these models as they require much more widespread expression, rather than a single region of interest (5).

     Imaging Probe Implantation

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 imaging probe into the brain where the GECI is expressed. There are three types of probes (optical cannula, cortical window, GRIN lens) that are used for in vivo calcium imaging. The probe you select is dependent on two factors:

  1. Do you require single-cell resolution imaging?
  2. Will you be imaging in a deep or surface brain region?

Optical cannulas enable light to be delivered and collected from the brain. These probes are used in Fiber Photometry (this will be discussed in the next post) experiments. Due to their design, optical cannulas are only capable of collecting one 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. Another bonus is optical cannulas are the least invasive surgery because of the compact design, which damages minimal tissue.

In contrast, cortical windows replace a large portion of the skull with a glass window. Neuroscientists employ cortical windows when imaging a large cortical region on the surface of the brain. Cortical windows provide access to the cortex for single-cell resolution recordings.

Lastly, a GRIN lens is a microendoscopic probe that can be implanted in the brain to image deep regions of the brain (up to 8mm) with single-cell resolution. GRIN lens differ in lengths, enabling neuroscientists to image shallow to deep brain regions. To minimize tissue damage, GRIN lenses are typically limited in diameter (0.5mm – 1mm). The GRIN lens diameter restricts the field of view, and thus, imaging with GRIN lenses usually provides a relatively small field of view, especially compared to cortical windows.

Equipment Components

Now that you have all biological components setup, including optimal GECI expression and imaging probe implantation, you need equipment to record fluorescent signals from the brain of a freely-behaving animal.

The systems currently available for in vivo calcium imaging (which we will discuss in the next post) are comprised of three main components:

  1. Coupling between the imaging probe and imaging device
  2. Light source and filter set
  3. Imaging device

      Coupling Between Imaging Probe and Imaging Device

A fluorescent signal is emitted from the GECI and this is transmitted through the imaging probe. But how can signal be collected?

First, you need a coupling between the imaging probe, light source, and imaging device. This coupling enables illumination of the GECI in the brain through the imaging probe and transmission of the emission signal to the camera. Depending on the calcium imaging system design, a coupling may be achieved via an imaging fiber, or an optical fiber, or the system (such as a miniscope) may be directly mounted onto the head of the animal.

     Light Source and Filter Set

Coupling enables you to illuminate and collect fluorescent signals from the brain. GECIs function by generating fluorescence signals, such that they have an excitation and emission spectrum (6). 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 in vivo calcium imaging since low optical power is required. However, if a larger region of interest is being illuminated, a higher-power laser may be required. There is a balance between too little and too much power: not getting enough signal and 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 and transmission of the correct emission signal to the imaging device.

     Imaging Device

Lastly, you need to collect and analyze fluorescent signals from the brain. This is made possible using an imaging device. Three types of imaging devices are used for in vivo imaging systems: 1) scientific camera, 2) PMT, and 3) photodetector. Which imaging device used is somewhat dependent on the calcium imaging system. If you’re interested in learning more about the differences between imaging devices, this is a helpful article.

Successful in vivo calcium imaging is 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.

Next Post

What systems are available for in vivo calcium imaging?


    1. Daigle, TL et al. (2018). A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell, 174(20), 465-480.
    2. Scott, BB et al. (2018). Imaging cortical dynamics in GCaMP transgenic rats with a head-mounted widefield macroscope. Neuron, 100(5), 1045-1058.
    3. Kondo, T (2018). Calcium transient dynamics of neural ensemble in the primary motor cortex of naturally behaving monkeys. Cell Reports, 24(8), 2191-2195.
    4. Resendez, SL et al. (2016). Visualization of cortical, subcortical, and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nature Protocols, 11(3), 566-597.
    5. Dana, H et al. (2014). Thy1-GCaMP6 Transgenic mice for neuronal population imaging in vivo. PLOS One.
    6. Grienberger, C & Konnerth A. (2012). Imaging calcium in neurons. Neuron, 73(5), 862-885.
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