Applications Leica LMD6500 & LMD7000

Leica Laser Microdissection Application Notes

Application Notes include short descriptions of research experiments.

Please convince yourself on versatility of Leica LMD Laser Microdissection system.

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The Morbus Parkinson Puzzle

Single-cell Analysis After Laser Microdissection

After Morbus (M.) Alzheimer, M. Parkinson is the second most common progressive neurodegenerative disease. Before the first symptoms manifest themselves, up to 70 percent of dopamine-releasing neurons in the mid-brain have already died. Dr. biol. hum. Falk Schlaudraff from the Molecular Neurophysiology team at the Institute of Applied Physiology of the University of Ulm used modern laser microdissection methods to isolate and analyze cells from post mortem tissue specimens taken from M. Parkinson patients in order to gain molecular insight into the disease.

Dr. Schlaudraff, what is the current state of M. Parkinson research?
A characteristic sign of M. Parkinson is the deterioration of dopaminergic neurons in the mid-brain, specifically in the substantia nigra (SN, black substance). Different causes and forms of this disease have been identified. In the case of the genetic familial form, for example, it has been possible to identify various genes that have a causal influence for M. Parkinson. How-ever, we still don’t know whether all the relevant genes have been identified, or exactly how they contribute to pathogenesis. There are several theories on how the disease originates. M. Parkinson can be compared to a puzzle. We have already found many of the pieces and can put some of them together, but we don’t know what the whole picture looks like. We haven’t worked out the significance of some of the pieces yet, neither do we know how many pieces of the puzzle we still have to find before we can present a full picture of M. Parkinson and actually understand the disease.

What was the focus of your research?
I concentrated on gene expression analysis of individual dopaminergic midbrain neurons of the substantia nigra. These cells selectively degenerate as the M. Parkinson disease progresses. Once a patient notices the cardinal symptoms of M. Parkinson, such as the resting tremor that is a characteristic of this disease, more than 70 percent of dopaminergic SN neurons have already died. One of the aims of my research was to develop optimized qPCR (quantitative polymerase chain reaction) assays. These enable valid comparison of the gene expression of promising gene candidates in individual neurons from human post mortem tissue of M. Parkinson patients with the gene expression of the same neurons from healthy control subjects. We developed a qPCR-based platform that could be used to isolate individual cells from native tissue and obtain highly comparable gene expression analyses. Our analysis, for example the RNA quality of the specimens, showed that the quality of the results is not influenced by the staining process and laser microdissection.

Why are dopaminergic neurons in substantia nigra particularly affected?
Analyses of the different post mortem specimens are like snapshots in a specific stage of M. Parkinson. I can compare these snapshots with each other. But I can’t use these specimens to detect whether a changed gene expression is the result or the cause of the disease.

What other results did you obtain from your research?
I detected an orchestrated change of gene expression in the selective dopaminergic neurons of M. Parkinson patients. This change affects genes that are involved in the regulation of the dopamine metabolism as well as genes that code for ion channels. We  showed, for example, a higher expression of several genes involv-ed in the synthesis and provision of dopamine in the surviving dopaminergic neurons. We also looked at various gene expression patterns of ion channels that regulate the activity of the dopaminergic neurons. Here too, we noticed a change in the expression of some of the examined genes of M. Parkinson patients.

What do you use laser microdissection for?
In the last few years there have been many studies comparing complete tissue specimens of substantia nigra of M. Parkinson patients with healthy tissue. However, this comparison is misleading, as in these patients, 70 percent of the neurons that are obviously involved in the disease are already degenerated at its onset. Also, the composition and the sectioning of the examined brain tissue are extremely heterogeneous. So the “tissue” approach we have adopted up to now is like comparing apples with pears.

We wanted to selectively view the midbrain dopaminergic neurons that are involved in the pathogenic process and used laser microdissection for validated comparison at the single-cell level. This technique makes it possible to accurately cut individual dopaminergic neurons out of complex tissue, without contact or contamination, and analyze the gene expression in individual cells. The most prevalent type of tissue in the brain is supporting tissue: Glial cells are ten to 50 times more common than the neurons we are interested in. Without laser microdissection, it would be almost impossible to clearly characterize the relatively rare nerve cells on a molecular level; they would not be distinguishable from background noise.

The analysis of single cells frequently leads to different results from those obtained from a complete tissue examination. Studies have shown that the expression of certain microRNAs is changed in the tissue of M. Parkinson patients. We followed up these statements and at first we were able to confirm the results for the whole tissue. However, we also exam-ined microdissected cells in parallel. Here we found that the microRNA expression is not changed on a single cell level. This tissue artifact was detected with the aid of laser microdissection.

What are the benefits of the laser microdissection system you use?
We chose the Leica Microsystems LMD system, which enables contact-free dissection of single cells or, if necessary, larger areas of tissue. The dissected material is captured in the cap of a tube and can be processed immediately. Leica Microsystems also gave us excellent support. Their technical service provided swift, comprehensive, and reliable answers to all sorts of questions.

What developments do you expect?
At present, there are no tests for diagnosing M. Parkinson at an early stage, and there are many variants of the disease and diseases with similar symptoms. A certain percentage of M. Parkinson cases are therefore diagnosed wrongly or not diagnosed at all. The prerequisite for successful treatment of M. Parkinson would be effective early diagnosis. If the disease could be diagnosed at an initial stage in which the neurons are just beginning to degenerate, it might be possible to prevent progressive neuron degenera-tion so that the disease does not break out at all. The identification of biomarkers in blood or cerebral fluid is currently a major research focus. There are genes that are not only expressed in the brain, but ubiquitously – in all cells. If the expression of these genes were changed in the dopaminergic neurons of an M. Parkinson case, it would be possible to examine more easily accessible tissue for diagnostic purposes. The first steps have been taken, but there’s still a long way to go before such a test can be implemented, and further long-term studies need to be conducted.

What projects are you working on at the moment?
After obtaining my Ph.D. in July 2010, I started work-ing in an R&D department of an industrial company, where I develop systems for analyzing nucleic acid.

Slides for Laser Microdissection

LMD applications require special slides, which are specifically designed to free the dissectate from the section.

For this reason, tissue sections to be microdissected are placed on a UV-absorbing membrane.

Upon the laser action, the membrane is vaporized, which also vaporizes the tissue in the cut line. Since the membrane is not adhered to the slide, the dissectate (with the attached specimen) drops into the cap of the microcentrifuge tube. Leica Microsystems offers high-quality membrane slides that provide high collection rates for laser microdissection.

There are three different membrane slides to choose from:

• PEN (polyethylene naphthalate)
• PET (polyethylene terephthalate)
• POL (polyester)

Membranes are mounted on regular glass slides (PEN), on steel frames (PET/POL), or on the bottom of special Petri dishes for cell cultivation. Depending on tissue type, thickness, and inspection method, one slide type may work better for a certain application. Therefore it is advisable to test different combinations of membranes and slide supports for the best results. Membrane combinations are summarized in a Table, click link above "Table Slides and Petri dishes".

Glass PEN foil slide (upper image on top) and the frame slide (upper image bottom).
Description: The membrane is mounted on the upper side of the slide (the side with the frosted end for marking). Since the membrane is glued on the edges in thecentral part of the slide, there is a space of 1 µm between the glass and membrane. The slides must be handled with care to prevent damage to the membrane, this will impede the laser action.

Petri dish for cell cultivation for LMD (right image).
Description: The Petri dish is placed on the holder for firm mounting on the microscope stage. Since the PEN membrane is perforated, the cells are perfectly supplied with oxygen. For better attachment and growth of cells, the membrane can be coated with poly-Lysine or collagen prior to seeding.

The frame slide (upper image on top) and the frame support (upper image on bottom).
Description of the frame slide: The front side of the slide contains the engraved Leica logo. Please note that only the back side of the slide (the side opposite the Leica logo) is foil mounted, and only this side can be used for mounting tissue sections.
Description of the frame support: The Plexiglas frame support contains the lifted surface (clear area) that fits exactly into the cavity or the front side of the frame slide. Not shown, the membrane slide must be turned over to be mounted onto the frame support.

Important: Please note that sterilizing slides by autoclaving or UV-treatment does not guarantee complete destruction of RNases. For RNA preparation, especially from a single cell (corresponds in average to 10-15 pg of total RNA) or from a small number of cells, RNase-free certified slides are recommended (see Table 1). Or alternatively, regular glass slides can be pretreated with reagents such as RNase Zap® (Ambion).

Citation list for Leica LMD Laser Microdissection

Citation list for Leica LMD Laser Microdissection

Please click here to open the pdf file with the citation list for Leica LMD Laser Microdissection.

Leica Microsystems - QIAGEN

One Solution – Two Competent Partners

Leica is a leading supplier of high precision optical microscopes and related instruments, and provides efficient tools for all the steps required for laser microdissection of tissue samples.
QIAGEN has a worldwide reputation for its technologies for nucleic acid purification, and provides optimized reagents and protocols for purification and analysis of nucleic acids from dissected samples.

The agreement between QIAGEN and Leica combines the expertise of two leading companies to bring you complete solutions for analyzing nucleic acids from laser microdissected specimens.

Leica Qiagen Brochure

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Reliable instruments for infiltration, embedding and sectioning

Prepare tissue sections by using a variety of sectioning techniques. Choose the appropriate Leica instrument for your application: cut frozen tissue with a cryostat, fixed and paraffinembedded tissues using the Leica RM22 family of rotary microtomes, or native, unfrozen tissue under physiological conditions with a Leica VT1200/1200 S vibrating microtome.

Efficient staining of tissue sections

Stain your fixed sections and visualize cell structures at high resolutions. Staining workstations from Leica allow several staining protocols to run simultaneously, and can be programmed to satisfy customized requirements. Built-in filters reduce exposure to hazardous fumes, and the staining process can be monitored through a transparent lid.

Experience a New Level of Speed

Safely and accurately, capture single cell(s) and tissue regions of any shape or size. The Leica LMD7000 allows you to easily collect samples with a fast, precise, contamination-free method - simply by gravity.
The Leica LMD7000 is a fully automated laser microdissection system based on the Leica DM6000 B. This automation allows you to harvest one or more cell types from a single slide directly into one or more PCR tubes.

Excise cells or metaphase chromosomes from a wide range of samples, including paraffin-embedded sections, frozen sections, blood smears, and stained and immunolabeled specimens.
The Leica LMD7000, was developed combining automated upright microscope architecture, three-dimensional optical control of the dissecting laser beam and the dissected area, non-contact tissue sampling and motorized post-dissection handling.

Efficient purification of DNA, RNA, miRNA, and proteins

Purify analytes from laser-microdissected tissue sections using QIAGEN® Sample Technologies that are specially designed to process very small amounts of starting material.
Efficient purification of DNA and RNA from laser-microdissected fresh-frozen tissues is achieved using the QIAamp® DNA Micro Kit (cat. no. 56304) and RNeasy® Plus Micro Kit (cat. no. 74034), espectively.

For lasermicrodissected formalin-fixed paraffin-embedded (FFPE) tissues, the QIAamp DNA FFPE Tissue Kit (cat. no. 56404), RNeasy FFPE Kit (cat. no. 74404), and miRNeasy FFPE Kit (cat. no. 217404) maximize recovery of usable DNA, RNA, and miRNA, respectively. Full-length proteins can also be extracted from whole FFPE tissue sections using the Qproteom^TM FFPE Tissue Kit (cat. no. 37623).

Highly specific and sensitive amplification for detailed characterization of nucleic acids

Amplify the nucleic acids purified from laser-microdissected tissue sections using QIAGEN Assay Technologies, which are ideally suited for very small amounts of starting material. Highly specific hot-start PCR solutions such as HotStarTaq® Plus DNA Polymerase (cat. no. 203603) and the QIAGEN Multiplex PCR Kit (cat. no. 206143) facilitate fast and successful molecular analysis of DNA.
The QuantiTect® Reverse Transcription Kit (cat. no. 205311) is optimized for cDNA synthesis with integrated genomic DNA removal from very small amounts of RNA (10 pg to 1 μg). Quantitative, real-time PCR and RT-PCR with QuantiTect Multiplex PCR and RT-PCR Kits (cat. nos. 204543 and 204643) allow analysis of multiple targets (2–4) in 1 reaction vessel on any commercially available real-time PCR cycler. All QIAGEN PCR and RT-PCR products include optimized buffers that enhance PCR specificity and minimize optimization of reaction parameters, enabling fast and successful results at the first attempt.