View source for Electrophoretic Tissue Clearing

Electrophoretic tissue clearing (ETC) is one of [[Sample Clearing#Clearing methods and strategies|two lipid clearing methods]] for the hydrogel-embedded tissue samples.  After completing the initial [[Sample Clearing#Clearing solution wash|clearing solution wash]] for two days to rinse out the excess hydrogel monomers, the sample can be placed in an ETC chamber to quickly remove the majority of the lipids and other unattached biomolecules from the embedded tissue sample.

==Methodology==
[[File: ETC_washing.png|thumb|500px|ETC applies an electric field to actively diffuse micelles through the tissue]]

The principles of tissue clearing are the same for both [[Passive Clearing#Methodology|passive clearing]] and ETC.  Both methods rely on SDS micelles to diffuse into the tissue, collect fatty lipids and other unattached biomolecules, and carry them out of the sample, leaving behind a hydrogel-tissue hybrid that is visibly transparent.

Passive diffusion of the micelles is a very slow process, and ETC accelerates it by taking advantage of the ionic charge on the SDS micelles.  When placed in an electric field, the negatively-charged micelles will actively travel from one electrode (where they are repelled) to the other electrode (where they are attracted).  Thus, applying an electric field across the hydrogel-embedded tissue sample in clearing solution stimulates active transport of the micelles in and out of the tissue.  Active micelle transport via electrophoresis significantly increases the speed of tissue clearing by orders of magnitude, such that a whole tissue sample like a mouse brain can clear in a matter of days instead of the weeks it would take to clear passively.

As observed when [[Passive Clearing#Methodology|clearing passively]], lipid removal during ETC causes the tissue sample to swell to a larger size.  Unlike passive clearing, though, ETC clears the whole sample at the same time, so the sample may not clear from the outside inwards but instead appear opaque throughout clearing and then become see-through all at once.

==Advantages and Disadvantages==
===Advantages===
*Fast process - almost complete clearing in days vs. weeks or months of passive clearing
*Can be combined with passive clearing

===Disadvantages===
*Requires [[Supplies#ETC set-up supplies|additional equipment]] for set-up and ETC chamber
*Small risk of tissue damage - burning or melting can occur if sample is exposed directly to electrodes or to chamber overheating
*Tissue yellowing occurs over time - may affect imaging penetration depth

==ETC set-up==
The ETC set-up is designed to provide fast, efficient clearing from active micelle transport through the hydrogel-embedded tissue while controlling the unavoidable by-products of electrophoresis that could be damaging to the tissue sample.  The [[Supplies#ETC set-up supplies|equipment]] described in the [[Supplies|supply list]] for setting up the ETC can easily be modified or replaced with alternatives that perform similar functions.

[[File: ETC_setup_schematic.png|thumb|500px|Schematic of the ETC components and solution flow during clearing]]
===ETC components===

====Components in ETC set-up:====
*''Circulator'' - controls clearing solution flow rate and temperature (could be replaced by liquid pump and temperature regulator)
*''Buffer filter'' - filters out any large particles in the solution to prevent them from diffusing through the tissue sample
*''ETC chamber'' - holds the tissue sample and platinum electrodes
*''Power supply'' - creates an electric field across the sample in the ETC chamber
*''Tubing and stopcocks'' - connect components and allow control of flow around each component

====Order of components====
About 4-5 L of clearing solution is placed into the circulator.  Tubing should be connected so that the clearing solution exiting the circulator flows through the buffer filter first to remove large particles from the solution.  The clearing solution then enters the ETC chamber through the inlet on the bottom of the chamber and flows through the outlet on the top.  After exiting the ETC chamber, the clearing solution flows back to the circulator which heats or cools the solution to maintain a set temperature.  Once circulation is established, the power supply is connected to the electrodes attached to the ETC chamber to supply an electric field across the tissue sample.  Plastic tubing connects all the ETC components for clearing solution circulation, and stopcocks are placed on either side of the buffer filter and ETC chamber so that flow can easily be stopped to unhook or check on each component without having to drain the solution from the entire system.

Circulation of the clearing solution through the different ETC components is especially important for preventing the build-up of electrophoretic by-products inside the ETC chamber from damaging the tissue sample.  These by-products include gas bubbles, acid, and most notably, heat.  By circulating a large volume of clearing solution through the chamber, the by-products are dissipated and prevented from accumulating inside the chamber and, more importantly, inside the tissue sample itself.  The order of the components is also designed to aid in protecting the tissue sample from damage.  The clearing solution flows through the buffer filter before entering the ETC chamber to block any large particles from penetrating the sample.  Within the ETC chamber, clearing solution flows from the bottom to the top to push gas bubbles up and out of the chamber and thus impede their accumulation within the sample.  The power supply is the last component to be hooked up and the first to be turned off as it supplies the heat and other by-products to the chamber.  It is necessary to ensure that clearing solution circulation is running before introducing an electric field into the chamber to avoid the risk of damage to the tissue.

===Variable settings===
ETC supports quick and efficient clearing of the tissue, but also produces certain by-products that could be damaging to the tissue sample and need to be neutralized.  Noticeable by-products from the electrophoresis of the clearing solution include:  gas bubbles, an acidic compound, tissue yellowing, black deposit on the electrodes, and most significantly, heat.  Some ETC settings are adjustable and allow control over both the rate of clearing and by-product maintenance.

====Flow rate====
As mentioned above, circulation of the clearing solution through the ETC chamber is extremely important in dissipating the build-up of electrophoretic by-products, specifically accumulation of gas bubbles, acid, and heat.  Of these by-products, heat accumulation poses the most serious risk of significant damage to the tissue sample in the ETC chamber.  The flow rate needs to be high enough to prevent heat build-up inside the chamber.  Certain perfusion pumps, for example, may be too weak for this task.  Subsequently, flow rates that are too high should also be avoided because they could lead to significant bubble accumulation from the circulating detergent or overly forceful pushing on the tissue sample in the chamber.

'''''Recommended flow rate:  1-2 L/min'''''

====Temperature====
In general, incubating the tissue sample in clearing solution at higher temperatures (37&deg;C and above) promotes faster tissue clearing.  However, heat build-up inside the ETC chamber is a serious concern as overheating could lead to significant tissue damage, even melting or burning the sample.  To ensure sample safety, it is recommended to keep the temperature at a lower setting and use increased clearing times or additional passive clearing before or after ETC.

'''''Recommended temperature setting:  37&deg;C'''''

====Voltage====
Similar to temperature, higher voltage settings promote faster lipid clearing, but lower voltage settings are safer for the tissue sample.  The voltage to the platinum electrodes is what creates the electric field inside the chamber as well as all the unwanted by-products.  As such, increased voltage leads to increased by-products, most noticeably heat and acid compounds.  The accumulation of either of these by-products inside the ETC chamber could lead to significant tissue damage.  Excess heat production needs to be counteracted with higher flow rates and good temperature regulation, even solution cooling, while acid accumulation will require replacing the clearing buffer solution more often, even in the middle of an ETC run.  Generally, for the safety of the tissue sample, it is recommended to keep the voltage setting in a lower range and run ETC for longer (or add passive clearing before or after ETC).

'''''Recommended voltage setting:  10-30 V'''''

[[File: Sample_in_ETC_chamber_above_view.png|thumb|400px|Overhead view of a tissue sample inserted between the electrodes of the ETC chamber]]

===Sample insertion===
When setting up ETC, the sample is placed in the ETC chamber between the two platinum electrodes.  The ETC chamber is then connected to the other components via plastic tubing, with a stopcock placed on either side of the chamber to allow temporary flow disruption while checking on the sample without having to drain the whole system.

====Sample holder====
The sample holder should be constructed out of insulating (non-conductive) materials that will not melt under the heat conditions inside the chamber.  It should also be porous enough to support good circulation of the clearing solution through the tissue sample and holder.  Suggested sample holders that meet this criteria and fit inside the [[Chamber Construction|constructed ETC chamber]] include cell strainers, paraffin tissue cassettes, or plastic mesh sleeves.  Additional [[Supplies#ETC chamber supplies|plastic mesh]] is placed on either side of the sample holder to prevent the sample from touching the electrodes during clearing, and thus avoid sample burning.

====Sample positioning====
Although still effective when the sample is allowed to freely move inside the sample holder, lipid clearing via ETC will go faster when the tissue sample is held in place inside the chamber.  The SDS micelles are actively transported in one direction (from one electrode to the other) across the sample, so clearing is more efficient when lipids are not being transferred back through areas of the tissue that have already cleared.  One way to hold a tissue sample, such as a whole mouse brain, in place inside the sample holder is to first place it inside an expandable plastic mesh sleeve that will prevent the sample from flipping directions inside the holder.  Take note when placing the sample into the ETC chamber which side is facing toward the anode and which side is facing towards the cathode so that if the sample is removed during the ETC run it can be placed back in the same orientation.  Also make sure that the polarity of the electrodes remains the same throughout the ETC run (it may help to label the electrodes on the outside of the chamber).  Alternatively, ensure that the sample positioning is switched in conjunction with changes to the electrode polarity.

The sample holder should be positioned relatively tightly between the platinum electrodes to allow minimal flow of solution in the areas surrounding the sample holder.  If there is too much space between the electrodes and the sample, the electric field will flow around the sample and not through it, leading to significantly slower clearing.  Extra plastic mesh can be placed in void spaces inside the chamber where needed to keep the sample holder in place between the electrodes.  Double check that the electrodes are as symmetric as possible so that an even electric field is applied across the sample.

==Running ETC==
===Starting up===
The following steps should be performed when starting up the ETC system for tissue clearing:
#Hook up all ETC components and add clearing solution to the circulator
#Place the hydrogel-embedded tissue sample in the ETC chamber and close the chamber tightly
#Start clearing solution circulation to initiate flow through all the components and check for leaking
#Connect the electrodes to power supply
#Turn on power supply to apply a voltage across the chamber electrodes

Because it creates electrophoretic by-products, especially heat, inside the sample chamber, the power supply is the last component to be connected and turned on, only after clearing solution circulation is established.  The power supply is also the first thing to turn off and disconnect before stopping circulation to remove the sample from the chamber or check on any of the components.

Once ETC is up and running, it can run continuously with little required maintenance.  A few steps are necessary, though, to prevent by-product accumulation from negatively affecting the tissue sample and/or clearing speed.

===Check pH routinely===
Acid accumulation in the clearing solution could damage the tissue sample and also disrupt the structure of the ionic micelles, thereby negating their ability to clear lipids from the tissue.  The presence of boric acid in the [[Solutions#Clearing Solution|clearing solution]] helps to buffer the acid build-up, but it can only slow, and not counteract, its effect on the pH of the circulating clearing solution.  To keep the SDS micelles intact, it is important to keep the pH level above 7.  It is recommended to regularly check the clearing solution pH (once or twice a day) and change the clearing solution when the pH dips into the 7.5-8 range.  Always use fresh clearing solution when replacing the circulating buffer (do not rebalance the pH in the old buffer using sodium hydroxide).

Some variables in the ETC set-up and settings may increase clearing solution degradation due to acid accumulation, thereby necessitating replacement of the clearing solution more often.  Such variables include higher voltage settings (40-50 V) and multiple chambers connected in parallel to one another (same volume of clearing solution is exposed to more electrophoresis in total than with only one chamber).  

Lowering the voltage setting applied to the ETC chamber will decrease the rate of clearing solution degradation.  If the ETC chamber is running at a low enough voltage such that acid accumulation does not show significant effects on the pH, it is recommended to replace the clearing solution after every 1-2 samples anyways.  Clearing is always more efficient with a fresh batch of clearing solution.

===Clean platinum wires routinely===
Over time, as the ETC chamber is used, black deposits will develop on one of the platinum electrodes inside the chamber.  The deposits accumulate as the chamber is used more and more.  Eventually the deposits will start washing onto the surface of the tissue sample, rendering the clearing process useless if the surface is covered in black deposits.  To avoid this outcome, the platinum electrodes need to be cleaned off every 2-3 days of use, even if it means interrupting ETC clearing in the middle of a run to do so.

====Methods for cleaning the electrodes====
The following methods have shown to be effective in cleaning off the black deposits from the platinum electrodes and preventing their transfer onto the surface of the tissue sample.

*'''Physical rubbing''' - Use a microfiber cloth and tarnish remover to physically clean off electrodes every 2-3 days of use.  The sample should be removed (place in 50 mL clearing solution in a conical tube) and the chamber should be drained and dried before cleaning.  After physical rubbing the electrodes may still not look clean, but enough of the black deposits will have been polished off the surface of the electrode to prevent accumulation on the tissue sample surface.

*'''Reverse electrode polarity''' - Every 2-3 days of use, remove the sample from the ETC chamber and place in 50 mL of clearing solution in a conical tube.  Reverse the polarity of the electrodes and run ETC with an empty chamber for 20-30 minutes to clean off the black deposits from the tarnished electrode.  Afterwards, place the sample back in the chamber and reconnect the electrodes to the power supply in their original polarity.  Be careful not to run ETC for too long with reversed polarity or a black deposit will develop on the opposite electrode.  This method has been shown to work really well when starting with a new ETC chamber (unused platinum wires), but it has not proven viable for used chambers that have already developed black deposits on the electrodes.
**''Note:'' If needed, a good time to replace the clearing solution in the ETC system is right after cleaning the electrodes.  The black deposits washed off from the electrodes will enter into the clearing solution and should be collected in the buffer filter.

===ETC duration===
The duration of ETC clearing is dependent on several factors including the individual set-up, equipment, settings, and user preference.  The clearing speed is highly dependent on the ETC settings, especially temperature and voltage.  To avoid tissue yellowing that can occur with extended periods of ETC, the user may prefer to use ETC for a shorter duration of time [[Sample Clearing#Clearing strategies|in combination with passive clearing]] before or afterwards.  This strategy provides the benefit of quickly clearing the majority of lipids from a tissue sample without exposing the sample too long to the negative effects of ETC.

==ETC completion==
To check the tissue sample for complete lipid clearing, place it in a conical tube of clearing solution and hold the container up to the light.  The sample will swell to a larger size during clearing.  Although the sample may still look a little cloudy, clearing is complete when the tissue is see-through across its entire span, at which point it can be placed in [[Sample Clearing#PBST buffer wash after clearing|PBST buffer for the next step in CLARITY processing]].

In contrast to [[Passive Clearing#Completion point|passive clearing]], where the tissue starts to show transparency on the outside of the sample first, the active transport of micelles through the sample during ETC often causes the sample to look entirely opaque until it suddenly looks entirely clear.  If the sample still looks opaque after several days of ETC, it may be mostly clear but have just enough residual lipids remaining to block light penetration.  Placing the sample in clearing solution for [[Passive Clearing|passive clearing]] several days after ETC should help to remove the remaining lipids.