Laser microdissection
MCB
1st April, 2013
Purpose
To allow us to utilise material that cannot be
obtained by other methods
To select only the cells or tissue that we want
Overview of LMD
• Excise tissue (cells) from tissue
• Dissection under microscope
• Exclude contaminating cells/materials
• Many potential tissue sources
• Accurate discrimination (staining, hi-mag)
Example of fine selection
Honaas et al. Functional genomics of a generalist parasitic plant: Laser microdissection of host-parasite interface
reveals host-specific patterns of parasite gene expression. BMC Plant Biology 2013, 13:9
Downstream applications
• QPCR, RNA-seq, microarray for gene
expression analysis
• Protein isolation
• DNA sequencing for tissue specific sequences
• More detail in the next laboratory presentation
Technical overview
• Attach tissue to membrane (PEN, etc.)
• Dissect out desired tissue with laser
• LMD is not the same as LCM.
Laser Capture
Microdissection
Emmert-Buck et al., 1996
The microscope
Microscope software
Laser power source
Microscope
X-Y and
objective
controls
Sectioning the tissue
A
B
C
Figure 1. Laser microdissection enables profiling of gene expression at different stages of leaf development.
Morphogenetic primordia (A, <750μm) were excised separately from maturing primordia (B, >750μm) and
from the shoot apical meristem (remaining in C). In addition to classification of primordia by length, the area
of the tissue excised is recorded. Tissues for each stage were pooled for RNA extraction using an Arcturus
Paradise kit. Randomly primed reverse transcription has yielded cDNA for quantitative PCR comparison of
development-associated candidate gene expression. Satisfactory yields were obtained from approximately 300
10μm tissue sections.
Harvest tissue
✓
Fix/preserve tissue ✓
Mount tissue on
membrane
Plant shoot tips – mature leaf and stem, young leaf and SAM
3:1 ethanol:acetic acid
Leica PEN membrane slides
(Heat block)
Deparaffinise
Stain material for
identification
Ethanol/ xylene; 100% ethanol; 95% ethanol; 70% ethanol
E.g. eosin, haematoxylin
Dissect tissue
Genomics Facility LMD 6000
Downstream
processing
RNA, DNA, protein, carbohydrates, etc.
Protocol-specific decisions
• Tissue type, quality and quantity
• Fixation method
- Paraformaldehyde; acetone; ethanol/ethanol:acetic acid;
methacarn (methanol, chloroform and acetic acid)
- Affects morphology and downstream processes
• Cryogenics or paraffin embedding
- Preservation of molecules or morphology
• Sectioning
- Section thickness; arrangement, pooling
• Additional steps
- RiboAmp?
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References
Leica LMD home - http://www.leica-microsystems.com/products/light-microscopes/life-science-research/lasermicrodissection/
Leica LMD preparation protocol - http://www.unige.ch/medecine/bioimaging/equipment/microscopes/protocoles.pdf
Fixation methods - Kim, J.-O., Kim, H.-N., Hwang, M.-H., Shin, H.-I., Kim, S.-Y., Park, R.-W., Park, E.-Y., Kim, I.-S., Van
Wijnen, A.J., Stein, J.L., Lian, J.B., Stein, G.S., Choi, J.-Y.
Differential Gene Expression Analysis Using Paraffin-Embedded Tissues after Laser Microdissection
(2003) Journal of Cellular Biochemistry, 90 (5), pp. 998-1006.
Plant tissue fixation - Kerk, N.M., Ceserani, T., Lorraine Tausta, S., Sussex, I.M., Nelson, T.M. Laser capture microdissection
of cells from plant tissues (2003) Plant Physiology, 132 (1), pp. 27-35.
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Example - Honaas, L.A., Wafula, E.K., Yang, Z., Der, J.P., Wickett, N.J., Altman, N.S., Taylor, C.G., Yoder, J.I., Timko, M.P.,
Westwood, J.H., dePamphilis, C.W. Functional genomics of a generalist parasitic plant: Laser microdissection of hostparasite interface reveals host-specific patterns of parasite gene expression (2013) BMC Plant Biology, p. 9. Article in Press.
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Arcturus Paradise kit manual - http://www.excilone.com/client/document/manuel-paradise_344.pdf
LCM method - Laser Capture Microdissection for Analysis of Gene Expression in Formalin-Fixed Paraffin-Embedded
Tissue. Ru Jiang, Rona S. Scott, and Lindsey M. Hutt-Fletcher
Overview with notes for hepatology, protein, RNA and DNA - Laser Microdissection Microscopy and its Applications in
Molecular Biology. Ahlam Mustafa, Ragai R Mitry and Alberto Quaglia, 2010
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Acknowledgements
Great thanks to Vijay Nadella at the Genomics Facility (5th floor, Porter Hall).
Useful discussions were had with Dr Faik and with Loren Honaas from Penn. State.