TITLE: Refractive index adjusted imaging medium: High RI Iodixanol, Iohexol - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare imaging medium for Saccharomyces cerevisiae with high refractive index. This medium is optimized for fluorescence imaging by the reduction of auto-fluorescence through an abundance of Adenine [1] and the repression of of the Met-promoted pp7- CP expression [2].

[BEFORE_START]
Have the following prepared:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 ml ddH2O

Methionine 200x 50 ml:
Concentration: 17.12 g/l
mix 856 mg into 50 ml ddH2O

Iodixanol crystals:
Preparation: 
transfer 40 ml Iodixanol (Optiprep) solution into a 50 ml centrifugation tube
place the tube opened into an oven set to 40°C
through evaporation the refractive index of the Iodixanol solution rises:
 
 
 

[STEPS]
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1. Compound medium for autoclave
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
4. The medium can be store at the bench for 2 to 3 months.
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
3. When the medium cooled down to around 80 °C add 3 mL sterile Glucose 20%.
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.7. Add and resolve Iodixanol (~Optiprep) crystals.
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.6. Add 1.4 mLddH2O.
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.5. Add 201.6 mg Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB).
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.4. Add 57.6 mg SC-XX powder. 
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.1. 
 

Fill a 50 ml flask with 25 mLOptiPrep.

Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
4. 
 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
3. When the medium cooled down to around 80 °C add 3 mL sterile Glucose 20%.
SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.6. Add Iohexol (Histodenz) powder.
 


 
 
 

 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.5. Add 1.5 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).


SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.4. Add 3 mL SC-XX 10x solution.
 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of 30ml high refractive index imaging medium with Iodixanol or Iohexol
1.1. Fill a 50 ml flask with 21.9 mLddH2O
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of high refractive index imaging medium with Iodixanol or Iohexol
1.8. Measure the refractive index with a refractometer. If the RI measured is below your target RI, repeat step 1.7  until the targeted RI is reached.