How To Extract Cells From Tissues Using Laser Capture Microscopy
Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.
So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National Cancer Institute by the Liotta group in the 1990’s, LCM involved placing a specialized, sticky film over a tissue section, observing the sample, followed by illumination of the target cells by a laser. This laser activates the sticky film, which in turn binds to the cells. This film can be lifted off and used to prepare samples for downstream applications.
The Pubmed search for ‘Laser Capture Microdissection’, clearly indicates that this technique is continuing to grow, powered in part, but the advances in genomics analysis.
Figure 1: Increase in publication using LCM. Data from Pubmed.
Today, there are a variety of different and unique methods for performing LCM. The major differences among the methods are the type of lasers used (UV or IR), what is moving (the stage or the laser) and how the cells get attached to the membrane.
Besides the original method for capturing the tissue, vendors have come up with novel solutions. In the Carl Zeiss PALM system, after the tissue of interest has been cut using a UV laser, a second pulse of the laser is used to propel the cut tissue towards a stick membrane, as shown in Figure 2 from the Carl Zeiss website.
Figure 2: The Carl Zeiss LCM approach – sometimes called Laser Microdissection Pressure Catapulting (LMPC)
Leica takes a whole different approach, and after the sample is cut, it allows gravity to drop the sample to the capture tube (figure 3)
Figure 3: The Leica approach using gravity to isolate the cut cells.
The power of LCM lies in its ability to isolate cells by extracting the right ones, proves useful in downstream applications. With the advances in sequencing and proteomics techniques, the small sample sizes are not an impediment in procuring high quality results . Additionally, in the case of RNA based research, care about the levels of RNAse in the tissue sample is imperative. This handy chart from Thermo-Fisher is helpful while planning the experiment as it highlights the relative levels of RNAse in different mouse tissues.
Figure 4:Relative levels of RNAse in murine tissues
Stringent care is needed while handling cells with high levels of RNAse activity. Tricks such as keeping everything very cold, avoiding long aqueous steps and dehydrating the sample before isolation of the RNA helps such experiments.
The first obvious step before delving into the world of LCM is to review the literature for your specific tissue of interest. Each cell type warrants a different optimal recovery approach for the downstream application. These tips may include the type of staining to be performed, or the best way to prepare the sample (FFPE or OCT embedding for example), and the potential new techniques that would be useful to adopt.
For example, one issue with RNA-seq technologies was overcome by a technique that Foley and co-workers introduced, Smart3SEQ. This technique was designed to address the ability to get gene expression patterns from very low total RNA samples. This was tested on the LCM samples after extracting it from FFPE prepared tissues, especially those that have been archived. The authors used a sample that was a year old in these experiments.
Figure 5:Image from Foley and Coworkers showing the single cell extracted using LCM
This sample was a ductal carcinoma in situ (DCIS) and the researchers isolated both bulk cells (~100 to 500 cells) and 10 single cells from the DCIS and some stromal macrophages adjacent to the DCIS. When the results between the bulk and single cells were compared, the data showed a clear picture that the single cell captures were able to recapitulate the results from the bulk captures (Figure 6 C). Additionally, a tSNE plot of the data clearly demonstrated that some extracting of the single cells that showed a gene expression pattern similar to the macrophage cells; clearly clustered with the macrophages (Figure 6D).
Figure 6: Taken from Foley et al., figure 4 in their paper
The heterogeneity found in the bulk tissue samples was only resolved at the single cell level, confirming the power of LCM in extracting cells in a spatial context.
The power of next generation sequencing coupled with the ability to measure expression patterns down to the single cell level offers researchers an amazing window to look at cell behavior. For solid tumors and tissues where cell-cell interactions can be important, using traditional flow cytometry methods are not useful as that information is lost. The use of LCM provides the researcher with the ability to accurately isolate cells, in a phenotypically defined manner, to take advantage of these techniques and enhance our understanding of biology.
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