Feb 06, 2025

Public workspaceRefractive Index Matching - EasyIndex

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx965kg8j/v1
Refractive Index Matching - EasyIndex
  • Holly Myers1,
  • Daphne Toglia2
  • 1Allen Institute for Neural Dynamics;
  • 2Boston University
  • Allen Institute for Neural Dynamics
Holly Myers
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx965kg8j/v1
Protocol Citation: Holly Myers, Daphne Toglia 2025. Refractive Index Matching - EasyIndex. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx965kg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 05, 2023
Last Modified: February 06, 2025
Protocol Integer ID: 81480
Keywords: Index matching, EasyIndex, LifeCanvas, Whole mouse brain delipidation, same refractive index as lifecanvas easyindex, refractive index matching, easyindex this protocol, same refractive index, lifecanvas easyindex, easyindex agarose embedding protocol, easyindex, index matching protocol, smartspim, mouse brains on smartspim, lifecanvas technologies full active pipeline protocol, imaging
Abstract
This protocol is modified from the Index Matching protocol found in the LifeCanvas Technologies Full Active Pipeline Protocol (see references). At the end of this process, the specimen should have the same refractive index as LifeCanvas EasyIndex, allowing for lightsheet imaging on the SmartSPIM. Refer to the following protocols to prepare for imaging: Embedding Protocol - SmartSPIM. EasyIndex Agarose Embedding Protocol, SmartSPIM setup and alignment, and Imaging cleared mouse brains on SmartSPIM.
Materials
Reagents:

ReagentLifeCanvas EasyIndexCatalog # EI-Z1001
ReagentNuclease-free water (e.g. MilliQ or HPLC grade water)

MaterialsProduct number
Serological pipets, 25mLFisher Scientific, 13-675-30
50mL high clarity conical centrifuge tubeFisher Scientific, 14-959-49A
Serological pipet fillerThemoFisher, 9501
OvenThermoFisher, 51028063
Aluminum foilAmazon, B074NB5CDZ
ShakerOHAUS, 404P87
Metal spatulaCole Parmer, ux-06287-07
Petri dishThermoFisher, 150460

Recipes:

50% EasyIndex for 1 Mouse Brain:

Using serological pipet to measure 10mL Nuclease free water into 50mL conical tube. Add 10mL LifeCanvas EasyIndex to container. Place upright on shaker at low speed for Duration00:05:00 to mix.

ReagentsVolumes
LifeCanvas EasyIndex10mL
Nuclease free water10mL


Troubleshooting
Safety warnings
Wear PPE while performing the steps of this protocol: gloves, eye protection, lab coat.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
This protocol is intended for a brain that has already been delipidated. See protocol Whole Mouse Brain Delipidation - LifeCanvas Active for preceding steps.

To allow time for troubleshooting (step 5), it may be beneficial to begin this protocol at the start of day.
LifeCanvas Index Matching
2d 0h 30m
Shake bottle of LifeCanvas EasyIndex well to homogenize the solution. Let the bottle sit for Duration00:30:00 to allow any bubbles to settle.
30m
Using a serological pipet, create 20mL of 50% EasyIndex by measuring 10mL of nuclease free water and 10mL of LifeCanvas EasyIndex into a 50mL falcon tube. Place upright on shaker at low speed for Duration00:05:00 to mix.

Add one delipidated whole mouse brain specimen into the tube and make sure specimen is completely submerged in solution. A long-handled spatula may be used to transfer the mouse brain into the tube. Seal tube tightly to prevent evaporation and cover in aluminum foil to protect from light. Incubate for Duration24:00:00 at Temperature45 °C on shaker at slow speed.
Note
The 50% EasyIndex solution may be stored at TemperatureRoom temperature if needed.


1d 0h 5m
Remove the 50% EasyIndex solution from the tube containing the specimen by using a long-handled spatula to scoop the brain out of the tube and pouring out the 50% EasyIndex solution from the tube. Replace the brain into the tube. Using a serological pipet, measure 20mL LifeCanvas EasyIndex into the tube and incubate for Duration24:00:00 in oven at Temperature45 °C on shaker at slow speed.
1d
Assessing for Successful Index Matching
22h
Remove tube with brain specimen and assess brain for successful index matching. The brain may be temporarily immersed in EasyIndex in a clear petri dish for evaluation.

Note
If processing a batch of multiple brains at once, assess each brain individually, as there may be variance in clearing between brains in the same batch.

Successful index matching:
When fully immersed in EasyIndex solution, the brain should be completely transparent. It should be possible to view numbers, letters, or fine lines on a grid through the brain without warping.

If the brain successfully index matched, proceed to step 6.


Example of a successfully index matched brain. Note that you can see the lines of the grid and read the number "23" through the brain when it is immersed in a dish of EasyIndex.


Not successfully index matched yet:
When fully immersed in EasyIndex solution, some opaque, white, or partially transparent structures may be visible inside the brain. It may be difficult to view images or lines through the brain without warping.

If the brain does not look successfully index matched yet, proceed to step 5.

Example of an unsuccessfully index matched brain. Note that you cannot clearly read the number "27" through the brain when it is immersed in a dish of EasyIndex. This brain could use the additional index matching step.

Place tube with the EasyIndex solution and brain specimen back into the Temperature45 °C oven on shaker at low speed for an additional Duration06:00:00 and assess brain specimen for transparency again (step 4).

If brain specimen is not yet completely transparent, remove the EasyIndex solution from the tube and replace with 20mL fresh EasyIndex. Replace specimen in Temperature45 °C oven for at least Duration16:00:00 on shaker at low speed.

This additional step should maximize index matching between the brain and the EasyIndex solution as much as possible. Proceed to step 6.

22h
The brain specimen is now ready for embedding, if desired. See protocol Whole Brain Embedding - EasyIndex with 2% Agarose for more details.

Sample may be stored in EasyIndex for several months at TemperatureRoom temperature .

Protocol references
Download Full Active Pipeline Protocol SmartBatch+ v5.06 (4).pdfFull Active Pipeline Protocol SmartBatch+ v5.06 (4).pdf