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Laser Capture Microdissection: Get it Out!

Posted in: Microscopy and Imaging
Laser Capture Microdissection: Get it Out!

Imagine you are studying a very interesting new protein. You know in which cell type the protein is expressed, yet that specific cell type constitutes only a small minority among a large collection of other cells in the tissue. Examples are endothelial cells in tumors, macrophages within an organ or a specific structure in the brain.

Ultimately, you would like to understand how this protein affects gene expression, epigenetic modulation or protein expression and phosphorylation in that specific cell type. However, when you isolate total RNA or protein, the sample is overruled by all the other cell types in that tissue.

So what do you do?

The solution is laser capture microdissection: a technique that allows laser-assisted separation of miniature samples, as small as a single cell, out of complex tissues.

Laser microdissection has been successfully used to identify genes that are highly expressed in the vasculature of high grade glioma but are almost absent in low grade and normal brain vasculature1.

The potential of laser microdissection is also illustrated in a study which showed that the effect of an anti-inflammatory drug complex specifically targeted to diseased glomeruli in the kidney could only be observed when looking at the glomeruli itself2,3. Analysis of the kidney as a whole masked the effect of the drug complex, which would have lead to the wrong conclusion that the drug complex is ineffective4.

How does it work?

A laser is coupled to an inverted microscope and focused through the objective onto the sample plane. This creates an energy-rich spot where the energy is high enough to cut through histological tissue sections. The microdissected specimen is then collected into an eppendorff tube in a contact-free way without affecting biomolecular integrity.  The sample is then processed for downstream analyses, such as quantitative reverse-transcriptase PCR, microarray or protein array.

What do you have to do?

Laser microdissection requires histological sectioning of the tissue material onto specific membrane slides that are biochemically inert. These slides are available from laser dissection microscope manufacturers such as Leica and PALM/Carl Zeiss. The sections then have to be immunohistochemically or immunofluorescently stained to visualise the specific cell type that you are interested in. Identify your cells on the laser dissection microscope and the laser does the job. It’s as simple as that!

Advantages

  • Laser microdissection allows you to investigate cell types that are underrepresented in a tissue, or to restrict your analysis to only those cells that express a specific marker or have a specific microscopic appearance.
  • It gives you valuable information on ’in situ’ cell behavior. Your sample represents the situation as it is in real life, in real time, and among all the other cell types that constitute the whole organ or tumor. This is in strong contrast to in vitro cell culture, where you take cells out of their natural environment.
  • Information on the location of the cells of interest and their histological appearance is maintained.
  • Laser microdissection can be used on frozen or paraffin-embedded materials, such as (archived) patient material.
  • But the technique is not restricted to histological sections of fixed tissues. As cell culture dishes can also be inserted in the microscope, live cell imaging and selection belongs to the possibilities as well. Think of microdissecting a beating cardiomyocyte, or cells where other active processes can be followed in real time.

Disadvantages

  • It can be a lot of work. Especially if you have chosen a cell type which is quite rare in the tissue of interest, you will need to dissect several thousands, perhaps even millions of squared micrometers of tissue to obtain sufficient material for further analysis.
  • It is inevitable that tissue sections contain damaged (unusable) cells and some material might also be lost during further processing. This may limit the possibilities of downstream analyses.
  • The selection is not 100% contamination free. Even though you can quite precisely control where the laser will cut the tissue, you cannot avoid lasering out a small part of the surrounding tissue as well.  We therefore tend to speak of an ’enrichment’ rather than ’purification’. But with enrichments of up to 100-fold, you can clearly distinguish the signal of any underrepresented cell type in any tissue!

All in all, laser microdissection is a powerfull tool to create a molecular magnifying glass!

References

  1. Dieterich LC, Mellberg S, Langenkamp E, Zhang L, Zieba A, Salomäki H, Teichert M, Huang H, Edqvist PH, Kraus T, Augustin HG, Olofsson T, Larsson E, Söderberg O, Molema G, Pontén F, Georgii-Hemming P, Alafuzoff I, Dimberg A: Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFb2 in vascular abnormalization. J Pathol 2012; 228: 378-390.
  2. Asgeirsdóttir SA, Kamps JA, Bakker HI, Zwiers PJ, Heeringa P, van der Weide K, van Goor H, Petersen AH, Morselt H, Moorlag HE, Steenbergen E, Kallenberg CG, Molema G: Site-specific inhibition of glomerulonephritis progression by targeted delivery of dexamethasone to glomerular endothelium. Mol Pharm 2007; 72: 121-131.
  3. Asgeirsdóttir SA, Zwiers PJ, Morselt HW, Moorlag HE, Bakker HI, Heerina P, Kok JW, Kallenberg CG, Molema G, Kamps JA: Inhibition of proinflammatory genes in anti-GBM glomerulonephritis by targeted dexamethasone-loaded AbEsel liposomes. Am J Physiol Renal Physiol 2008; 294: F554-F561.
  4. Langenkamp E, Kamps JA, Mrug M, Verpoorte E, Niyaz Y, Horvatovich P, Bischoff R, Struijker-Boudier H, Molema G: Innovations in studying in vivo cell behavior and pharmacology in complex tissues – microvascular endothelial cells in the spotlight. Cell Tissue Res 2013, Sep 27.
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