Electrophoretic Tissue Clearing

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Electrophoretic tissue clearing (ETC) is one of two lipid clearing methods for the hydrogel-embedded tissue samples. After completing the initial 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.

Contents

Methodology

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ETC applies an electric field to actively diffuse micelles through the tissue

The principles of tissue clearing are the same for both 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 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 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 clear the hydrogel-embedded tissue sample quickly and efficiently while counteracting the unavoidable by-products of the electrophoresis process. The equipment described in the supply list for setting up the ETC can easily be modified or replaced with alternatives that perform similar functions.

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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.

clearing solution then flows back to circulator which heats or cools solution to maintain set temperature - power supply is connected to electrodes inside chamber - tubing connects all the components for clearing solution circulation - stopcocks placed on either side of buffer filter and ETC chamber so that flow can easily be stopped to unhook or check on each component without having to drain 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 entering the sample. Within the ETC chamber, clearing solution flows from the bottom to the top to push gas bubbles up an out of the chamber and thus impede their accumulation within the sample.

Variable settings

The ETC set-up is designed to provide fast, efficient clearing from active micelle transport through the tissue while controlling the by-products of electrophoresis that could be damaging to the tissue sample. Noticeable by-products from the electrophoresis of the clearing solution include: gas, an acidic compound, tissue yellowing, black deposit on the electrodes, heat. Some ETC settings are adjustable and allow control over both the rate of clearing and by-product maintenance.

Flow rate

-counteracts two by-products: gas bubbles and heat build-up in the chamber - transports bubbles to top and out of the chamber to prevent accumulation within the sample which would occur without clearing solution circulation - most importantly, dissipates heat and prevents chamber and tissue sample from overheating which could lead to significant damage even in just a short period of time

flow rate needs to be high enough to prevent heat build-up (certain perfusion pumps may be too weak for the task) but not so high that there is significant bubble accumulation from the circulating solution or that it is pushing too forcefully on the tissue sample in the chamber

Recommended flow rate: 1-2 L/min

Temperature

-higher temperatures (above 37°C) promote faster clearing - heat build-up inside the chamber is a concern though as it could lead to tissue damage (melting) - better to keep temperature at lower setting and increase clearing time for sample safety

Recommended temperature setting: 37°C

Voltage

-higher voltage promotes faster clearing, but lower voltage is safer for tissue sample - voltage to the electrodes is what creates all the by-products, so increased voltage also leads to increased by-products, most noticeably heat (need to counteract with higher flow rate and solution cooling) and acid compounds (need to counteract by replacing clearing solution buffer more often, possibly mid-run) - build-up of either of these by-products could lead to significant tissue damage - better to keep voltage in lower range and clear for longer to keep sample safe

Recommended voltage setting: 10-30 V

Sample insertion

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Overhead view of a tissue sample inserted between the electrodes of the ETC chamber

- when setting up ETC, sample is placed in ETC chamber between the two platinum electrodes - the ETC chamber is then connected via tubing and stopcocks to the other components - clearing solution circulation is turned on first to initiate flow through the chamber and check for leaks, then the electrodes are hooked up to the power supply and voltage is applied

Sample holder

-sample holder should be constructed out of insulating (non-conductive) materials that will not melt under the heat conditions of the chamber - show pictures (?) of suggested sample holders: cell strainer, parafin cartridge, plastic mesh sleeves

-additional plastic mesh is placed between the platinum electrode and the sample holder to prevent the sample from touching the electrodes and burning

Sample positioning

-clearing goes faster when sample is held in place (i.e. micelles always travel in the same direction so that lipids are not being transported back through areas that have already cleared) - use plastic mesh sleeve or sample holder to hold sample in place (show picture?) and take note when placing the sample into the chamber which side is facing toward the anode and which is facing toward the cathode (also make sure that the polarity of the electrodes remains the same or that the sample positioning is switched in conjunction with the electrode polarity)

-sample should be positioned tightly between the electrodes to allow little flow of solution around the sample (otherwise electric field will flow around sample and not through it, leading to significantly slower clearing) - plastic mesh can be used in the chamber where needed to keep the sample holder in place - double check that electrodes are as symmetric as possible so that an even electric field is being applied across the sample

Running ETC

Starting up

-hook up all ETC components -place sample in ETC chamber -start clearing solution circulation and check for leaking -hook up electrodes to power supply -turn on power supply - last component to hook up and turn on and first component to turn off

-once ETC is running, can run continuously with little required maintenance - a few steps are necessary though to prevent accumulation of electrophoresis by-products that can negatively affect the tissue sample and clearing speed

Avoid heat build-up in chamber

move this section elsewhere? reiterate settings?

Check pH routinely

-acid buildup could damage tissue and also disrupt the structure of the ionic micelles, ruining their clearing effects -keep pH level above 7 - change solution buffer when pH dips to 7.5-8 range - always use fresh buffer (do not rebalance pH in old buffer)

variables that increase buffer degradation, therefore need buffer replacement more often: higher voltage settings (40-50 V), multiple chambers set up in parallel (same volume of clearing solution is exposed to more electrophoresis in total than with only one chamber)

lower voltage setting to decrease rate of buffer degradation

if running at low enough voltage that acid buildup does not show significant effects on the pH, change buffer solution after every 1-2 samples anyways - clearing is more efficient with fresh batch of buffer

Clean platinum wires routinely

-black deposits develop on one electrode as ETC chamber is used - gets worse as chamber gets more used, eventually starts washing on to surface of the tissue, rendering the clearing process useless if surface is covered in opaque deposits

Methods for cleaning the electrodes

  • Physical rubbing - use microfiber cloth and tarnish remover to physically clean off electrodes every 2-3 days of use - only viable option for used chamber that has developed the black deposits - electrodes may not look clean, but enough is polished off the surface to prevent build up on the sample surface
  • Reverse electrode polarity - remove sample from chamber every 2-3 days of use, reverse the polarity of the electrodes and run ETC with empty chamber for 20-30 min to clean off electrode before placing sample back in chamber and returning electrode hook up polarity to normal - be careful not to run too long or black deposit will develop on the opposite electrode

ETC duration

- duration of ETC dependent on the set-up, equipment, settings, and user preference - clearing will go at different rates for different settings - might also want to use ETC for shorter durations of time in combination with passive clearing to benefit from faster clearing of a majority of lipids without accumulating negative effects of ETC - completion point similar to that for passive clearing, see-through sample, may not be entirely transparent

ETC completion

Visual check - what to expect (sample might still look opaque all the way through

Passive clearing - might be good afterwards to finish clearing residual lipids; can also be applied before ETC (link to strategies)

PBST buffer wash when sample is see-through