A long-standing objective in neuroscience has been elucidating how in vivo neural activity relates to sensory processing, behaviour, cognition, and cortical processing. Researchers have attempted to understand this relationship by developing a wide-range of all-optical tools for calcium imaging in freely-behaving animals.

The requirements for in vivo calcium imaging vary depending on imaging resolution, animal model, field of view, data collection, and brain region. With a varying degree of requirements, there are different calcium imaging tools available to fully understand the complex connection brain activity and function.

What systems are available for in vivo calcium imaging?

1. Fiber Photometry

Fiber photometry is an in vivo calcium imaging tool that detects average fluorescence intensity changes from population neural activity within a select region of a freely-behaving animal (Cui et al. 2014). An implanted optical cannula coupled to an optical fiber allows light to be delivered and retrieved from the brain. The acquired signal is then collected by an externally positioned imaging device (photodetector, PMT or camera).

Fiber photometry data acquisition is limited to population level activity with no cellular resolution to visualize individual neurons. The benefit of low cellular resolution is small data files, fast acquisition, and easy data interpretation, unlike other current calcium imaging tools. Thus, fiber photometry provides a low entry barrier for new labs wanting to adopt calcium imaging or perform exploratory research.

The light-weight design and less invasive surgeries make it possible to perform fiber photometry in multiple brain regions simultaneously. Multi-region fiber photometry is performed using a multi-fiber patch cord and imaged onto a camera for data acquisition (Kim et al. 2016). In comparison, a single region fiber photometry experiment uses a single patch cord is used and signal is captured using a photodetector or PMT.

The light-weight equipment required for fiber photometry helps extend the length of experiments and reduce extraneous factors (e.g. stress) allowing for more natural animal behavior to be observed during experiments. In addition, certain systems can be used with a rotary joint for freely-behaving experiments.

Fiber photometry is a useful tool that can provide us with a better understanding of low level circuitry in the brain. The simplistic design and data output of this tool provides a good starting point for in vivo calcium imaging.

Free eBook Download
In Vivo Calcium Imaging: The Ultimate Guide

2. Miniscope

A miniscope is a miniaturized microscope that mounts on the head of an animal to image neural activity in a freely-behaving animal (Ghosh et al. 2011). By coupling a miniscope to an implanted GRIN lens (deep brain) or a cortical window (cortex), you can image individual neurons in a  freely-behaving animal. The design of a miniscope is essentially the same as a one-photon microscope, comprised of the appropriate lenses, LED, filters, and camera.

The construction of the miniscope unlocked the ability to image the activity of thousands of individual neurons in freely-behaving animals (Ghosh et al. 2011). Freely-behaving capabilities are supported due to the reduced weight of the miniscope that has all the components integrated (~2g) into one system. The minscope provides a field of view that is determined by the size of the implanted GRIN lens (ranging 0.5mm to 1mm diameter) and the selection of GRIN lens will depend on the region of interest.

Recent developments in miniscope technology have enabled researchers to perform dual-colour imaging, wireless calcium imaging, and two-photon calcium imaging in freely-behaving animals (Zhong et al. 2017; Shuman et al. 2020).

The miniaturized design and all components being integrated on the head of the animal allows the animal to behave freely but partially constrains the possible integrated components. These include, low level cameras with low sensitivity and high noise not capable of high cellular imaging resolution, and the number of wavelengths they can illuminate is currently restricted to one or two. Thus, the capabilities of this system and the flexibility for future updates are currently limited.

Miniscopes have advanced our understanding of neural activity. This system can provide further insight into the activity of large neuronal populations for calcium imaging in freely-behaving animals.

3. Optical Fiberscope

The optical fiberscope, such as Mightex’s OASIS implant, is an all-optical system that enables single-cell resolution calcium imaging in freely-behaving animals using an imaging fiber. A removable imaging fiber, coupled with a GRIN lens implanted in the brain or cortical window, provides calcium imaging in the deep brain, cortex, or spinal cord of a freely-behaving animal.

The imaging fiber consists of thousands of individual micro-fibers to image hundreds of individual neurons in freely-behaving animals. An optical fiberscope can also be used to perform  population level imaging, identical to fiber photometry. This enables researchers to begin experiments with fiber photometry and later delve deeper using single-cell calcium imaging. Like a miniscope, the optical fiberscope’s field of view is determined by the size of the implanted GRIN lens.

A wide-range of calcium imaging applications can be executed with the optical fiberscope, such as dual-colour imaging and multi-region calcium imaging. The optical fiberscope is the only system that can image multiple brain regions with cellular-resolution — to view individual cells — in a freely-behaving animal.

The flexible imaging fiber and weight of the head-mounted fixture is very low (as little as 0.7g). And, all the electronics are off the head of the animal, compared to a miniscope. Thus, the length of experiments can be extended and extraneous factors (e.g. stress) can be reduced, allowing for more natural animal behavior to be observed. To add to this, the recent implementation of an intricate rotary system enables better freely-behaving experiments.

A vital benefit of the optical fiberscope is the unique flexible design that is scalable and reconfigurable, making it a generic calcium imaging and stimulation platform that can be adapted for different applications, unlike many other single-purpose systems. Two illumination paths allow researchers to attach multiple wide-field and/or targeted light sources with different wavelengths, and to insert different optical filters (e.g. dichroics etc.) suitable for different imaging and/or illumination needs. In addition, this system is compatible with high-quality scientific cameras for capturing better quality images (e.g. with better signal-to-noise ratios and better linearity) for data analysis.

The optical fiberscope is an ideal flexible tool to help understand how single-cell interactions are involved in advanced brain functions, which is not possible with other current technology.


System Comparison Table

Next Post

Can Optogenetics be Integrated with In Vivo Calcium Imaging?


Related Products for In Vivo Calcium Imaging

Mightex's OASIS Implant for
Freely-Behaving Calcium Imaging
The OASIS Implant is designed for freely-behaving cellular-resolution imaging
and optogenetics to probe complex neuronal networks.
  • Simultaneous Cellular-Resolution Imaging and Optogenetics
  • Multi-Region Investigation
  • Reconfigurable Platform
  • High-Quality Imaging with Scientific Cameras



Free eBook Download
In Vivo Calcium Imaging: The Ultimate Guide