TITLE: Sampling leaf tissue for analysis of NPQ Relaxation using  Technologica Chlorophyll Fluorescence Imager Data.
AUTHORS: Lynn Doran

[DESCRIPTION]
Sampling leaf tissue for analysis of NPQ Relaxation using  Technologica Chlorophyll Fluorescence Imager Data.

[STEPS]
SECTION: Leaf Tissue Sampling
1. Cut #1 Whatman filter paper into small squares that are just bigger than the well on a 24-well culture plate.
SECTION: Leaf Tissue Sampling
5. Using a #2 Humboldt cork borer, take leaf tissue samples from each plot for each technical replicate.  Hold the leaf flat against the rubber stopper, press the cork borer through the leaf into the stopper and twist.  Try to use the most recently fully expanded leaf with mature chloroplasts and limited bug damage.  Avoid sampling over the midrib.

 

If taking multiple technical replicates from a plot, multiple leaf discs can be sampled simultaneously and will continue to move up the cork borer.
 

SECTION: Leaf Tissue Sampling
4. Label the lid of the plate and the side of the plate with the plots sampled.  
 

SECTION: Leaf Tissue Sampling
6. Using a long cotton swab, or spatula, push the leaf discs out of the cork borer into the appropriate well of the 24 well plate.  All technical replicates can be in the same well but each plot (biological replicate) must go in it's own well.

 

SECTION: Leaf Tissue Sampling
7. When a complete 24 well plate has been sampled, place the lid on.  Store the plate out of direct sunlight at room temperature. 

Typically a bag, box, or empty cooler (no ice) works well.
SECTION: Leaf Tissue Sampling
8. When all plates have been collected, return to lab.
SECTION: Leaf Tissue Sampling
9. Using tweezers, transfer each leaf disc to an individual well in a 96 well plate.  The top  of the leaf disc should be facing down on to the bottom of the 96 well plate.

Handle the leaf discs gently.  Damage to the tissue, i.e. being poked with tweezers, will affect chlorophyll fluorescence analysis.

 

 

SECTION: Leaf Tissue Sampling
10. Dip a nasal aspirator filter halfway into water and then immediately dab on a paper towel.  Insert filter in the well on top of the leaf disc.  Gently press the nasal aspirator filter all the way to the bottom of the well, ensure that the leaf disc is flat on the bottom of the 96-well plate and that the damp filter is touching the leaf disc.
 

 

SECTION: Leaf Tissue Sampling
12. When all wells have leaf discs and sponges inserted, place the lid on the plate.  Tape the top right corner to help orient the plate in the dark for imaging.

 

 

SECTION: Leaf Tissue Sampling
13. Parafilm the lid to the plate by wrapping the edges with parafilm.

 

SECTION: Leaf Tissue Sampling
14. Wrap the plate in aluminum foil and write the plot IDs and plate ID on the aluminum foil.

 

SECTION: Leaf Tissue Sampling
15. Place in a dark box or cabinet for a minimum of 1 hour, but preferably overnight before imaging and analyzing for chlorophyll fluorescence.

 

SECTION: Leaf Tissue Sampling
2. Use the #7 cork borer to push two small squares of #1 Whatman filter paper into each well of the 24-well culture plate.
 


SECTION: Leaf Tissue Sampling
3. Wet the filter paper using tap water.  The filter paper should be thoroughly soaked but there should not be standing water left in the well.
 

SECTION: Leaf Tissue Sampling
11. If using the PSI Fluorcam, the camera field of view from the third shelf can accommodate one and a third 96 well plates for maximum throughput. Continue plating leaf discs through row C of the second 96 well plate.  Replace both lids and use lab tape on the top to attach the two plates in the correct order.  Ensure that none of the tape covers the wells on the bottom.