Correlation of Phenotype and Genotype – microscopic samples and their biomolecular backgrounds

Laser microdissection – the method of choice for single cell or sub-cellular isolation and subsequent high throughput analysis

April 20, 2017

Seeing is believing, this is what makes microscopes, imaging systems, or box-type imagers so important for researchers in many different fields like pathology research, cancer research, neuroscience, and developmental research. Correlation of phenotype (what you can see) and genotype (genetic background) is highly desired but often biased by whole tissue approaches as no real solution is available to reliably separate the single cells of interest and surrounding tissue. Thus, results of DNA mutation analysis (sequencing), gene expression profiling (quantitative real-time PCR, qPCR, microarray, digital PCR), next generation sequencing (NGS) or mass spectrometry (MS) approaches can give mixed results as no pure starting material for the biomolecular methods of choice is available.

Challenges to isolate and preserve single cells in liquid suspension

However, the technology landscape for isolation, extraction and other purification methods to obtain such pure starting material like single cells are available, e.g. FACS (fluorescence-activated cell scanning) or Fluidigm devices for single cell auto-preparations. The challenge for these powerful high-throughput microfluidics technologies is to get the cells in suspension. This crucial need to get the tissue or cells into liquid suspension for the cell sorting systems might affect the molecular content especially for cells in a tissue context. To preserve the quality and molecular biology pattern of cells in a tissue context, which can be observed with a microscope or any other imaging solution, is highly recommended. Thus, a technology to somehow specifically select, extract, isolate, separate, puncture, suck, sort or simply cut out the desired single cell(s), cell clusters or distinct layers from a tissue section will help.

Punching out cells or areas of cell clusters might result in biased results as the structures under the visible surface might differ from what you can see on the top layer. Therefore, the best way to ensure getting reliable pure starting material is to disassemble a 3D tissue into serial sections and select from each serial sections the cells or areas of interest as starting material.

Electrophysiology: great technique, but not suitable to collect high amounts of single cells

Electrophysiology is a great example of such phenotype and genotype correlations. For example, signals from individual neurons in a vital slice can be measured under microscopic control. This neuron’s content (cytoplasma) or the cell body including the nucleus can be sucked into a special needle with buffer to preserve the content on a specific time point. This technique however is not applicable for high throughput applications where higher amounts of single cells are required (e.g. for mass spec) and also not applicable for human tissue as the patch clamp relies on vital brain slices from laboratory animals. Human tissue usually comes as cryo-preserved or FFPE (formalin-fixed, paraffin embedded) sample serving as irretrievably starting material for phenotype – genotype correlations. A clear need for a versatile method of various tissue types – dead or alive – is highly desired.

Laser microdissection: the method of choice to collect pure starting material

Such a technology exists, it is called Laser Microdissection (LMD) [1], or Laser Capture Microdissection (LCM), or Laser Assisted Microdissection (LAM). The initial invention was implemented on a Leitz Orthoplan far ahead of time [2]. Arcturus re-invented the technology and started to successfully commercialize it. However, ownership of Arcturus changed frequently: From Applied Biosystems to Life Technologies and to finally end with Thermo Scientific (in 2016), systems like the XT, the Veritas, Pixcell and Pixcell II(e) are still used to obtain enriched target tissue. PALM Lasertechnologies and MMI (Molecular Machines and Industries) followed with their inventions of the PALM Microbeam (nowadays owned by Zeiss) and the MMI CellCut (Plus) systems.

Figure 1: Laser microdissection is able to excise tiniest structures such as amyloid plaques. Image before and after excision. Courtesy of Carlo Condello, Department of Neurology, UCSF (USA)

Leica LMD systems: dissectate collection by gravity

Later, Leica Microsystems invented their own solution (AS LMD) which completely differed from the other solutions. The main differences were to use an upright instead of an inverted microscope as base stand and take advantage of the gravity to collect simple, quick and gently dissected microscopic ROIs (region of interest) contact- and contamination-free. On the other hand, the laser focus is guided over the sample like a microscope scalpel for cutting, instead of relaying on a fixed laser focus and stage movement for dissection (table saw principle). This gravity collection has a big advantage as it allows to connect this extraction technology of microscopic samples with state of the art analysis methods like liquid capillary systems for downstream analysis [3] like mass spec or any other microfluidics device.

Laser Microdissection: Cutting of sample

Laser microdissection applications

The Leica LMD systems are microscopes with integrated scissors to cut areas of interest as tiny as individual chromosomes up to wood sections of several hundred micrometer thickness. This microscope extraction tool is nowadays routinely used to separate tumor areas from surrounding healthy tissue for mutation analysis [4], individual neurons for gene expression analysis [5] and neuronal layers or plaques for proteomic analysis [6]. Beside these application fields many more LMD applications in developmental biology, clinical research, forensics, live cell culture, cloning and other research fields are common for the system. The extraction will work in different contrasting methods such as fluorescence, phase contrast and brightfield.