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-moving animals.

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

What systems are available for in vivo calcium imaging?

1. 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). With an implanted optical cannula coupled to an optical fiber, light is 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. However, 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, and the signal is captured using a photodetector or PMT.

The light-weight equipment required for fiber photometry helps to extend the length of experiments and reduce extraneous factors (e.g. stress), allowing for more natural animal behavior to be observed during experiments. Also, 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 provide a good starting point for in vivo calcium imaging.

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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 large populations of individual neurons in a freely-behaving animal. The design of a miniscope is essentially the same as a one-photon microscope, consisting of the appropriate lenses, LED, filters, and camera.

The development of the miniscope unlocked the ability to image the activity of thousands of individual neurons in freely-behaving animals (Ghosh et al. 2011). The ability to imagine in freely-behaving animals is supported by the miniaturization 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 from 0.5 to 1mm diameter), and the selection of the 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 (Zong et al. 2017; Shuman et al. 2020).

With a miniaturized design and all components being integrated on the head of the animal, the miniscope allows the animal to behave freely, but it also partially constrains the possible integrated components. These include low-level cameras with low sensitivity and high noise that are not capable of high-resolution imaging, 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 neural populations with 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 cellular-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, enabling the imaging 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 cellular-resolution 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 weight of the flexible imaging fiber and the head-mounted fixture is very low (as little as 0.7g). And, all the electronics are located off the head of the animal, compared to the 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 with the optical fiberscope. To add to this, the recent implementation of an intricate rotary system enables better freely-behaving experiments with the optical fiberscope.

A vital benefit of the optical fiberscope is the unique flexible design. This system 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) suitable for different imaging and/or illumination needs. Also, 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 a flexible tool designed to help understand how single-cell interactions are involved in advanced brain functions, which is not possible with other current technology.


System Comparison Table

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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