Due to the poor penetration of visible light into the brain, one challenge for optogenetic control strategies is to deliver light into deep structures (> 1-2 mm). This can be achieved rather conveniently using optical fibers. Optical fibers are a particular type of waveguide (see page fiber optics). A waveguide is basically a structure which guides the propagation of waves such as electromagnetic waves (in other words light). Waveguides conducting light (lightguides) can be of several types, but the most convenient and affordable lightguides for transmitting light on long distances are optical fibers.
Although fiberoptic technologies are already extremely advanced, mostly because of their intense use in telecommunications, fiberoptic light delivery into the brain has required some minor technical innovations. Amongst them was the possibility to deliver light in an unrestrained freely moving animal through a rotary fiberoptic connection (see page Fiberoptic Rotary Joints). Fiberoptic connectorization also had its deal of tweaking thanks to optogenetics, mostly toward miniaturization. This page is a review of currently available fiberoptic connectorization kits for small animals.
Light Delivery Through a Guide Cannula
Optical fibers can be guided into the brain through an implanted cannula. A protocol for light delivery through a guide cannula is available in . In this protocol, the extremity of an optical fiber is introduced into a smaller cannula (the internal cannula) which fits into the guide. The fiber + protective internal cannula assembly can be inserted and secured into the guide for the duration of an experiment and removed afterwards. A dummy internal cannula replaces the fiber + internal cannula while the animal is housed between experiments. Cannula sets can be purchased from Plastics One for approximately $13 for a cannula pair. This method has both advantages and disadvantages.
- Viral infection and light delivery are in register if the guide cannula is also used for virus injection.
- Repeated insertion and removal of the fiber/cannula assembly and dummy cannula can damage the tissue. In particular it is not unusual to observe a blood clot at the end of the guide even weeks after implantation. This might be caused by a negative pressure effect (= suction) happening when removing the dummy after leaving it in place for a while (days).
- Depending on the fiber diameter, the diameter of the guide (which has to accomodate the fiber + protective internal cannula) can become quite significant (> 500 µm).
- If special attention is not taken to cleaning and sterilization of the fiber/cannula assembly and dummy cannula, this approach significantly enhances the risks of post-operative infections.
- When combining optical stimulation and electrophysiology, the pressure created by inserting and removing the cannula can disturb the electrodes, changing their location relative to brain tissue.
Light Delivery Through an Implanted Optical Fiber
In this case, a small piece of optical fiber is permanently implanted into the brain over the region of interest. Light is delivered into this fiber (fiber A) by another fiber (fiber B) through a fiberoptic connector mounted onto the animal's head. This connector can be a miniaturized and/or simplified version of an existing fiberoptic connector, or any custom-built connector which is able to bring the cores of fibers A and B in close apposition with good precision and reproducibility. Different versions of such connectors are commercially available and detailed below. Like the previous approach, this approach also has its advantages and disadvantages.
- When compared with cannulated fiberoptic light delivery, this approach has the benefits of
- reducing the diameter of the implant (just the optical fiber penetrates the brain).
- reducing the chances of post-operative infection.
- limiting tissue damage to the implantation procedure.
- For some types of connectors, coupling/uncoupling of the implanted optical fiber to a light source (through an optical fiber) can easily be done while the animal is awake.
- This solution is probably more expensive than the previous one when using currently commercially available connectors ($60 per implant vs. under $20).
- Some light is necessarily lost at the fiberoptic coupling interface, although this can be limited by using a gel whose refractive index matches the one of the fiber core.
Ferrule Connectors (fiberoptic cannula)
This type of connector was initially referred to as the "mono fiberoptic cannula" in Doric Lenses' catalog. The term has gained acceptance since, although the word "cannula" can be confusing (see the first paragraph of this page about the method consisting in using a removable fiber through an implanted cannula - a method that somehow went out of use).
The implantable "fiberoptic cannula" consists of a segment of optical fiber mounted in a ferrule. Ferrules have been used for a long time in fiberoptic coupling but they were usually part of a larger assembly (example: the FC/PC connector). The part of the fiber protruding out of the ferrule is implanted in the brain, while the ferrule itself is anchored to the skull using dental cement. The ferrule can be connected to another ferrule using a sleeve made of zirconia. Ferrules can be made of metal or zirconia (beware: zirconia ferrule of the same batch usually have less variability in their outer diameter than metal ones - check with your suppliers to make sure that your ferrules fit tightly in the mating sleeve).
Commercial ferrule connectors
Ferrule connectors/Fiberoptic cannula can be purchased from Doric Lenses, which was the first to propose this type of connector in its catalog.
Thorlabs also proposes similar ferrule connectors/Fiberoptic cannula:
How to make custom ferrule connectors?
While "fiberoptic cannula" can be purchased from a few companies (see above paragraph), it is relatively simple and very cheap to make these assemblies in the lab. Steel or ceramic zirconia ferrules can be purchased in a variety of sizes from companies such as Precision Fiber Products and sleeves can be purchased to couple 1.25 mm or 2.5 mm ferrules. With practice, it is possible to create about 20 implantable ferrule/fiber assemblies in an hour, with the total material cost of ~$1 per assembly.
Briefly, a procedure for making implantable 1.25mm ferrules with 105/125 fiber is as follows (pictured at right):
- Strip several long segments of 105core/125cladding optical fiber.
- Cleave these segments into ~30mm sections (this exact length is not important but they should have good flat cleaves on both ends and be longer than 20mm).
- Slide each segment into a 1.25mm ceramic zirconia ferrule with a 126um inner bore, and push it all the way through until the bottom edge is flush with the bottom of the ferrule. It is best to set up about 20 of these at this step so they can all dry together.
- For each assembly, place a drop of 5 minute epoxy on the top part of the fiber, just above the ferrule.
- Push the fiber down until the desired length of fiber protrudes from below the ferrule, and the epoxy balls up at the top of the ferrule (take care not to let the epoxy get on the sides of the ferrule, or wipe it off if it does).
- Let everything dry for at least 15 minutes.
- Cleave off the excess fiber at the top of the ferrule and polish the ferrule using conventional methods.
- Test the power transmission by hooking it up to a laser and using a light meter. If the assembly exhibits bad light transmission it can often be improved with a little more polishing.
- Check also the detailed how-to for making these connectors in Sparta et al, 2012 and http://www.syntheticneurobiology.org/protocols/protocoldetail/35/9.
Dual-Fiber Connector (a.k.a. "Dual Fiber Cannula")
Doric Lenses has developed a dual fiber design, in which one ferrule contains two guide holes for fibers. This design also relies on an alignment hole to keep everything in the right orientation.
Miniature SMA Connector
Miniature SMA connectors are one of the smallest commercially available connectors. These benefit from a screw-in design, ensuring a secure fit.
This type of connector utilizes two small Neodymium magnets to secure the patchcord on the animal head. The receptacle contains the implantable optical fiber, a part made of titanium which contains a series of grooves to ensure good anchoring into the acrylic cement, and a ring magnet. The patchcord is terminated with a zirconia ferrule centered inside another ring magnet. A spacer glued onto the magnet is used to decrease the strength of the magnetic connection and make it compatible with the intended use (easy plugging/unplugging with minimal mechanical stress on the animal head).
Fiberoptic Light Guides with Diffuser
When the optical fiber is implanted in brain, its tip delivers narrow cone of light in front of the fiber. The angular spread of light coming from a fiber tip is determined by the numerical aperture (NA) of the fiber. For example, the optical fiber with 0.22 NA spreads the light within the apex angle of 25.4° in air . When implanted or immersed into body liquids, the angular spread of the light coming out of the fiber tip is reduced by the factor of 1.33 to 19.1°, where 1.33 is approximately the refractive index of the body liquids. As this angle is relatively small, some of the neurons of interest for optogenetics or electrophysiology experiments may not be illuminated.
To overcome this limitation, Doric Lenses has developped a diffuser layer for the tip of the fiber-optic to increase the angular light spread. Typically, fiber-optics with diffuser layer fill the 300° apex angle (solid angle > 3.7π [sr]) with light. This is over 74 times larger in terms of solid angle, when compared with 0.22 NA standard fiber-optic where the solid angle is 0.05π [sr]). In air, the light intensity falls-off gradually as the angle increases (Figure 1). Consequently, for the same amount of input light power, the fiber-optic with diffuser layer will illuminate larger area albeit with reduced illumination intensity. To estimate the illumination pattern in biological tissues, Doric Lenses team also measured the angular spread in water. Water modifies the light transmission at tip and yield to a significant larger output intensity than in air; total output power is 1.56 ± 0.05 X higher in water (mean ± S.D., N=7 tested fiber-optics). The resulting illumination pattern is maximum in front of the tip and intensity never drops under 20% of peak maximum over 300° (Figure 1 and 2). The intensity drops theoretically as 1/r² where r is the distance from fiber tip. Additionally, the loss induced by the diffuser layer are almost 50% of the input power in air yet drop around to 25% in water. Therefore, the diffusive tip enlarges the illumination area but needs higher illumination power.
Advantages of the diffuser layer
- light is spread into larger solid angle, over 70 X bigger than conventional optical fiber.
- several electrodes can be illuminated.
- the illumination pattern allows very efficient cross-section excitation.
- the rapid intensity drop useful for local stimulation; varying input power enables fine tuning of excitation reach.
- the wide illumination requires two order of magnitude stronger input power to obtain the same light intensity.
- the inherent loss of the diffuser:50% in air and 25% in water.
- the diffusing layer is sensitive to certain solvent as acetone. Though, It can be cleaned with isopropanol alcohol.
Hybrid fiberoptic cannulas (liquid + light delivery)
Led by requests from researchers at the Riken Brain Science Institute, Doric Lenses has designed a hybrid cannula with metal tube that guides the optical fiber and restricts liquid delivery to the zone around the fiber tip. This "hybrid" fiberoptic cannula connects to miniature fiberoptic connectors (see above).
- Doric Lenses.
- Doric Lenses' blog: "Optogenetics-at-Doric".
- Precision Fiber Products.
- White Paper: "All-Optical Deep Brain Imaging & Stimulation Tools for In Vivo Neuroscience" has provided an overview of the existing all-optical imaging and stimulation tools, and compared the advantages and disadvantages of these techniques.