RootflowRT v.2.8

 

Imaging in the Baskin Lab

 

Image Quality

The software’s ability to make accurate motion estimation is integrally dependent on image quality. Good images have a histogram that is spread out and there is good focus throughout the root.

 

Fairly Good Root Image:

Motion Mask Image

 

Resulting Velocity Profile

 

 

 

 

Very Poor Root Image:

Motion Mask Image

 

Resulting Velocity Profile

 

Imaging Procedures

1.     Plates grown on agar can be imaged either from the top using the dimple lids or from the back through the agar.

2.     If imaging Control roots, which will grow fairly fast, set the inchworm in Step mode to travel 400um. If imaging slow growing roots such as for IAA and 2,4-Dtreatments, then set the travel distance to 300um.

3.     The best way to obtain images that will yield the best data is to first get the best focus possible across the entire length of the root and then adjust the light source of the microscope to obtain the best spread of gray values from white to black. Note, if you are imaging through the agar, the image may appear to have too much contrast, just try your best.

4.     In Waheeda’s macros of Scion Image, <c> will initiate the taking of a stack initially set as stack ‘1’, ‘9’ frames ‘15’ seconds apart. These can be changed manually after hitting <c>.

5.     Also in Waheeda’s macros, <b> will take a background image for the stack number entered. Eg. Stack0003Back 0001. This is appropriate if the background mosaic method is being used.

 

Setting up a Root for Imaging using Scion Image

1.     Focus on the root tip so that the end of the root is about 2/3rds across the full screen. (Note, this is a rule of thumb that will have to be adjusted depending on how fast growing the root is and how speedily you set up the whole root to begin taking stacks. These factors will affect the position of the root tip by the time you are imaging it. The QC should be clearly visible and the root tip should take up over half of the frame for successful velocity analysis and mosaicing.)

2.     Loosen the inchworm holder and manually move the inchworm and stage so that it is making full contact with the stage and the root in the same position as in step 1. (Caution! The inchworm is delicate. Please be gentle when bringing it into contact with the stage. Do not bang the stage onto the inchworm. Be sure the stage is counterweighted before starting to work with the inchworm.)

3.     Reverse the inchworm in Step mode the appropriate travel distance until the root maturation zone. Adjust the stage so that it is making full contact with the inchworm during this process.

4.     Begin taking frames of the root stack using <c> macro by selecting LoadMacros in Scion Image and choosing Waheeda’s. This will be stack 1.

5.     Travel forward using the inchworm in Step mode and take stack 2. Repeat until the root tip stack is reached. If everything goes well, the root tip will not have grown out of frame by the time you reach it.