Workflows & Protocols: Plant Laser Microdissection

Report on a Leica LMD user workshop held in Brazil

June 06, 2016

During Leica workshops for LMD users in Brazil, hosted by the Federal University of Paraná/UFPR (UFPR) at the Centro de Energia Nuclear na Agricultura/USP (CENA), the power of laser microdissection using the Leica LMD systems was demonstrated.

One special focus was on plant dissection which needs a high laser power. Product Manager Dr. Falk Schlaudraff explained how to pepare plant samples for laser microdissection to use the Leica LMD system to dissect plant tissues (e.g. roots or leaves).

Why dissect plants?

Plants consist - like humans and animals - of different kinds of tissues which are of scientific interest in many areas like agriculture and nutrition. Plant roots for example or also leaves of the plant, the flowers and the wood of trees as well. Depending on the question behind it is necessary to identify particular substructures of a plant tissue and isolate this substructure to receive a clear picture of its molecular content.

Fig. 1: Introduction to the system for a user group. Plant dissection is a perfect example as the Leica LMD7 (successor of the Leica LMD7000) has a very powerful laser which allows to dissect plant leaves without any slide as specimen carrier and directly collect the dissectate via gravity.

Plant leaves are usually easily to collect and serve a perfect specimen for laser microdissection. Leaves can contain inclusion bodies or particular areas affected by a disease which are of interest for further investigation or for comparison with surrounding normal leave tissue. Native plant leaves without any special treatment or preparation could be directly supplied to laser microdissection with the Leica LMD7.

The laser is powerful enough to dissect a native leave even in low magnifications, the collection via gravity is of advantage as well as heavy dissectates fall down quickly after dissection without any additional force – quick and easy.

Fig. 2: A native leave without any preparation or treatment applied to the Leica LMD system

During the workshop customers dissected different types of roots, leaves, flowers, and grass on their own with the Leica LMD system after brief introduction. This process is very easy and fast, as no special specimen preparation nor are any expensive consumables required.

The LMD process can be compared to a scalpel dissection of the leave but in much smaller dimensions (µm range). After cutting with the laser scalpel the dissectate simply drops down into the collection vessel (e.g. a PCR tube cap) and is ready for downstream analysis (such as PCR, qPCR, mass spec etc.).

Preparing parts of plants for laser microdissection

To improve the flatness of leaves or similar strong tissues, the specimen could be placed between two frame slides (called “frame sandwich”) which allows a faster navigation with almost no refocus requirements. For such “frame sandwiches” it is crucial to only use frame slides, glass slides on top of such thick and hard specimen will flatten the specimen as well, but these will cause a high risk of LIGE (laser induced glass etching): the high laser power might scratch the glass and thus making it extremely difficult to cut as the laser power gets absorbed by the (etched) glass. For this reason frame slides are recommended for high laser power applications (plants incl. woods, bone, teeth etc.).

Fig. 3a: A leave with a tiny hole after laser microdissection with the Leica LMD7000 (predecessor of the Leica LMD7)
Fig. 3b: The hole in the leave as observed with the LMD system
Fig. 3c: The dissected piece of the leave in the tube cap

Preparing plant roots with fluorescent worms inside

Beside plant leaves some plant roots with fluorescent worms inside where of interest for some workshop attendees. For this special task the roots where first natively clamped between two frame slides and the laser settings were adjusted to allow cuts through the whole roots. However, the identification of the fluorescent worms inside the whole roots was not possible. To achieve plain cuts of the roots with the cryotome the procedure was as follows:

  1. First, some OCT (Optimal Cutting Temperature compound) on the sample holder was frozen in the cryotome at -35°C.
  2. Linear scratches were applied into the frozen OCT with a sterile needle.
  3. Into each scratch one root was placed lengthwise.
  4. The aligned roots in the scratches were covered with OCT and placed back into the cryotome at -35°C for 30 min.
  5. The cryotome was set to -20°C and the prepared OCT block with the aligned roots allowed to equilibrate for 45 min.
  6. The prepared OCT block with the aligned roots was cut into 35 µm sections and sections containing root tissue mounted on PPS steel frame slides.
  7. The sections were briefly dipped into Acetone (stored at -20°C) for fixation.
  8. The root sections were investigated within fluorescence mode with the Leica LMD system.
Fig. 4: Group dynamics: happy workshop customers educate each other and share ideas on diverse application options.

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