- Choose approximately 35 leaves from the drying oven (no more than the number of currently unused PVC caps), ideally all from the same sampling date.
- Scan each leaf at 300 dpi in color. Make sure there are standoffs on the scanner bed before closing the lid. Make sure all leaflets are separate and not on top of each other. Save the file with the unique leaf identifier in the format MMDDYY-NN in a folder on the Desktop named 'Scanned leaf images'.
- Cut a 1 cm x 1 cm section from each leaf using a razor blade. If the leaf is smaller, use the entire leaf. If any tissue is left, return it in the original envelope.
- Place this leaf section into a PVC cap. Cut off a 5 cm section of coffee stir-stick and write the leaf ID on it in pencil. Do not use pen!
- Dip the PVC cap, rounded side down, in PVC cement. Let it air-dry in the fume hood. This will seal the label and prevent dye from obscuring the text.
- Place the label stick rounded-end down in the correct PVC end cap.
- Mark the original leaf envelope with a large check mark and your name to indicate it has been used. Return all the envelopes to the drying oven.
- Begin the leaf clearing protocol with the leaves in PVC end caps. Once each leaf is immersed in NaOH at a stable warm temperature, you can leave. Make sure the container lid is closed!
These steps should take 3-4 hours! This is a good stopping point.
- Arrange to return 6-24 hours later to check on the leaves. Make sure the water level is high enough; add more water if necessary. Then change the NaOH bath if it has turned dark brown. Waste NaOH goes into the disposal container in the fume hood - use a jar or weigh-boat to transfer the liquid.
- Repeat the previous step until leaves are transparent; move them to DI water for storage or continue to the next step. Thick leaves may take several days.
These steps should take less than 30 minutes! This is a good stopping point.
- Following the clearing protocol through destaining and storage in pure ethanol. Reuse and store all solutions except colored destaining solution and water rinses. Water rinse can go down the drain; other chemicals need to go into the waste container.
This step should take 2-3 hours! This is a good stopping point.
- Follow the leaf clearing protocol for mounting in toluene and Pro-Texx. Label each slide with permanent marker with the leaf ID and the current date. Any waste can go in the trash.
- Store slides on cardboard sheets on a lab benchtop. Write down in the log book which slides you completed.
- Rinse PVC caps and tools in the sink.
These steps should take 2-3 hours! This is a good stopping point.
- Turn on the light-table. Make sure the surface is clean, or wipe it down with water and/or ethanol.
- Set the microscope magnification to 25 (so the 25 lines up with the indent on front side of the scope body). Set the microscope aperture to its widest setting (all the way to the left).
- Make sure the camera is set to mode P, ISO 100, mirror lockup on, 2-second timer, 0 exposure compensation, fluorescent white balance, neutral colors, RAW capture.
- Place a millimeter ruler on the light table. Focus roughly on this image.
- Turn on the camera-control software and enable live-view with zoom. Complete the focusing of the image.
- On the computer, make a folder in the 'Cleared leaf images' folder named with the current date. Store all images you take today in this subfolder.
- Take an image (remember you need to press the camera button, not the software button on the computer). After you have pressed the button to take the image, don't lean on the table or touch the scope so that vibrations and image fuzziness are minimized. Name this image with the current date and RULER (e.g. 092310RULER.CR2).
- Don't touch any of the magnification settings on the microscope! If you do, repeat the previous steps to calibrate the scale.
- Remove the ruler from the light table and instead place a leaf slide on the table.
- Focus on the slide and take an image. Remember to use live-view + zoom to ensure the sharpest possible image!
- Name this file with the leaf identifier (e.g. 060110-23.CR2).
- When you have imaged all the slides, turn off all the computer equipment and place the dust cover over the microscope. Return the slides to their cardboard sheet, but mark each completed slide with your initials.
These steps should take 2-3 hours! This is a good stopping point.