The first chapter in our Fundamentals Series on deep-tissue imaging referred to the importance of recording from behaving animals and discussed the typical challenges involved with this. Upcoming chapters will focus on specific methods. We intend to, very briefly:
- Introduce the basic principles behind the method
- List some of its main benefits and restrictions (with reference back to the challenges mentioned in the first chapter)
- Refer to existing commercially available systems and
- Look at the latest developments, and possible future trends
The first method and the topic of this second chapter is fiber photometry.
Fiber photometry is a well-established method, where light is projected down single or multimode optical fiber/s, either directly implanted or coupled to an implanted probe or cannula in the brain (Figure 1). This light can be used for activating optogenetic actuators and for exciting injected or genetically encoded reporters, typically Calcium indicators (GECIs). Emitted light from reporters can be transmitted back along the same excitation/activation fiber/s and separated from the excitation light with dichroic mirrors before detection by photomultiplier tubes (PMTs), photodiodes, an
Figure 1. Schematic of a basic fiber photometry setup
Benefits and Restrictions of Fiber Photometry
Fiber photometry in its simplest format (single fiber, light source, single photodiode) allows you to record from a population of neurons without the additional complication of spatial information. The optical components are widely available and standardized, and analogue output signals can be digitized with equipment commonly available in neuroscience laboratories. This means that it is relatively easy to generate data using this technique (5).
There are many variations on fiber photometry (see Recent and Future Developments section below and keep an eye out for next week’s chapter on the fiberscope). These lean toward more complexity, for example increased channel count, using a camera instead of a photodiode or PMT, adding a spectral unmixer and customizing the shape of the tissue-end of the fiber to achieve certain effects.
- Minimum head weight
Because no electronics are mounted on the head, the weight-load on the animals head is minimal (generally below 10g 1),
- Negligible heat generation
Again, because no electronics need to be on the head, no heat is generated near the tissue.
A simple photometry setup, consisting of the fiber and fiber mount, LED, suitable dicroics, bandpass filters and a detector (can be a fairly inexpensive photodiode), is relatively affordable.
Commercial solutions start around £8k, with more complex solutions around £20-30k.
- Potentially fast Acquisition Rate
Fast acquisition is possible, with analogue outputs from PMTs and photodiodes being digitized at the sampling frequency of the analogue-to-digital converter, often in the kHz range.
This is fast enough to detect action potentials, and may become very interesting with recent advances in genetically encoded voltage indicators (GEVIs) and following action potentials in fast-spiking neurons. GECIs have fast kinetics, but picking up signals from fast-spiking neurons is often problematic, with calcium still being present in the cytosol in between APs (5).
When using a CCD or CMOS camera as the sensor, frame rate is determined by the camera frame rate. Improvements in frame rates in sCMOS technology offer very fast acquisition speeds, often without much compromise in sensitivity.
PMTs and, to a lesser extent, photodiodes are sensitive to even single photon count levels. Since these signals are picked up from a large volume, even low activity levels can be detected with fiber photometry.
- Customizable fibers
The fiber can be custom-treated in a variety of ways (e.g. tethered, painted or etched), to achieve different optical and geometric qualities. Since fibers can be customized with readily available instruments to neuroscience labs, variables such as fiber taper or having detection windows etched along the fiber extend the range of experiments which can be performed (5, 7).
- Thin fiber implants reduce invasiveness
Fibers used for fiber photometry are typically between 240 and 480um (2). This is less than any of the other methods using implants (GRIN lenses, micro-lenses), which makes this method less invasive.
Thinner fibers can be used, but this often means a compromise in recording area, or lower signal levels.
With some experimental setups, the fiber implant is directly connected to the detection hardware. This poses a challenge in handling animals between chronic recordings, and is not allowed by some ethics authorities. This issue is addressed by disconnecting the animal between recordings, often by implanting a cannula and lowering the fiber into the cannula during recordings. It may be challenging to find the same brain area from one experiment to the next, since no spatial information is acquired.
An alternative is to have an implanted piece of fiber or GRIN, and mate the front end of the fiber with this probe on a high-precision head-mount. There are always extra losses in transmission efficiency in the coupling, but it offers the additional flexibility of using a lens with specific optical properties near the brain.
Recent publications circumvented the tether problem by developing wireless photometry modules, where microLEDs were combined with on-the-probe detectors and dichroic mirror components (2).
- Limited spatial information
The optical and geometric specifications of the detection tip of the probe and the excitation wavelength and intensity determine the area around the tip of the probe which will be activated or recorded from.
This volume can be approximately calculated (7), but besides this information, photometry systems offer no spatial information, with data originating from the population within this volume.
Several optics component companies offer parts for fiber photometry (e.g. Newport, Edmund Optics or Thorlabs), and it is relatively easy for labs with some technical proficiency to assemble complete systems. Several companies, like MCI-Neuroscience (contact us to discuss your requirements), NPI, Plexon and Doric offer turn-key solutions.
New and Future Developments
Building on recent developments in wireless photometry technology, wireless recordings co be further improved by integrating multi-colour excitation LEDs, with higher transmission frequency (currently 28Hz) and further improvements in weight and heat specifications (2).
More sophisticated methods for developing different fibers and treating fibers before implant may also lead to interesting developments, like the method in next week’s chapter, the optical fiberscope.
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