Experimental Set-up for Explant Imaging

The Tissue Slicers:

For explant imaging, samples must first be harvested and sliced using one of our two vibrating blade microtomes.  These tissue slicers produce accurate sample sectioning without the need for freezing.

The Leica Vibratome is a mostly automated system capable of live and fixed tissue section.  This instrument is also built to slice tissues either freely or embedded in agarose.  The set up allows sectioning of delicate tissues from very large samples (up to 33 x 40 x 15 mm).  

The Precisionary Compresstome is fully automated to section both fixed and live tissues with a thickness range of 4µm to 1000µm and an adjustable precision of 1µm.  This instrument is able to section samples with a maximum diameter of 12.5mm and length of 3mm. 

 

The Perfusion Chamber Kit:

 

 

 

a. Y Connector Setup
b. Post
c. Perfusion Chamber- check for broken coverslip
d. Needle (MUST be in kit)
e. Right Angle Clamp
f. Tubing, lots of it connected to In-Line Heater
g. Extra Perfusion Pump Hoses (not required, but nice)
h. In-Line Media Heater (cable connected)

 

The perfusion chamber on "Gen 3" in action!

(Hint: follow the yellow tube)

 

First, media is warmed in a water bath and infused with carbogen gas (the clear tube).  

Then, the prepared media is piped out and makes its way to the sliced sample.                                     

 

Next, the media passes through the in-line media heater (black cylinder connectded to the post and clamp) before it is delivered to the tissue, which is in an open chamber (the perfusion chamber) beneath the objective.  The stage heater (black and white cords) warm the perfusion chamber and help keep the slice alive.  A vacuum flask (not in view) is hooked up to the system (clear tube and Y-connector).  This allows a constant and fresh flow of carbogenated media to reach the living slice.  

 

 

Close up view of the perfusion chamber with a tumor slice directly beneath the objective.  The tissue slice is first adhered to a coverslip and then stuck to the bottom of the chamber, which is a glass slide.  The in-line heater (black cylinder) delivers fresh media to the slice, the stage heater (black and white wires) warm the chamber, and the clear tube aspirates the depleted media.

 

Side view of the set up showing the vacuum flask connected to the system  and collecting the old media.   

 

 

 

 

With the perfusion chamber kit completely set up, it is finally time to turn off the lights and image!