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. 

extracting

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. 

extracting

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.  

Concluding Remarks

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.  

To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Similar Articles

4 Critical Rules For Spectral Unmixing

4 Critical Rules For Spectral Unmixing

By: Tim Bushnell, PhD

Spectral unmixing is the mathematical process by which a spectrum is broken down into the abundances of the different fluorochromes that make up the observed spectrum. This was described in the paper by Novo et al., (2013), which presented a generalized model for spectral unmixing of flow cytometry data. Of course, like compensation in traditional fluorescent flow cytometry, there are important rules to observe regarding the controls that are used to unmix the sample. If you need a refresher on the rules for TFF compensation, you can read about them here.    This blog will discuss the generalized process of spectral unmixing…

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

By: Tim Bushnell, PhD

So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

How To Do Variant Calling From RNASeq NGS Data

How To Do Variant Calling From RNASeq NGS Data

By: Deepak Kumar, PhD

Developing variant calling and analysis pipelines for NGS sequenced data have become a norm in clinical labs. These pipelines include a strategic integration of several tools and techniques to identify molecular and structural variants. That eventually helps in the apt variant annotation and interpretation. This blog will delve into the concepts and intricacies of developing a “variant calling” pipeline using GATK. “Variant calling” can also be performed using tools other than GATK, such as FREEBAYES and SAMTOOLS.  In this blog, I will walk you through variant calling methods on Illumina germline RNASeq data. In the steps, wherever required, I will…

How small can you go? Flow cytometry of bacteria and viruses

How small can you go? Flow cytometry of bacteria and viruses

By: Tim Bushnell, PhD

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

By: Tim Bushnell, PhD

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

Understanding Clinical Trials And Drug Development As A Research Scientist

Understanding Clinical Trials And Drug Development As A Research Scientist

By: Deepak Kumar, PhD

Clinical trials are studies designed to test the novel methods of diagnosing and treating health conditions – by observing the outcomes of human subjects under experimental conditions.  These are interventional studies that are performed under stringent clinical laboratory settings. Contrariwise, non-interventional studies are performed outside the clinical trial settings that provide researchers an opportunity to monitor the effect of drugs in real-life situations. Non-interventional trials are also termed observational studies as they include post-marketing surveillance studies (PMS) and post-authorization safety studies (PASS). Clinical trials are preferred for testing newly developed drugs since interventional studies are conducted in a highly monitored…

How To Optimize Instrument Voltage For Flow Cytometry Experiments  (Part 3 Of 6)

How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

By: Tim Bushnell, PhD

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

How To Profile DNA And RNA Expression Using Next Generation Sequencing (Part-2)

How To Profile DNA And RNA Expression Using Next Generation Sequencing (Part-2)

By: Deepak Kumar, PhD

In the first blog of this series, we explored the power of sequencing the genome at various levels. We also dealt with how the characterization of the RNA expression levels helps us to understand the changes at the genome level. These changes impact the downstream expression of the target genes. In this blog, we will explore how NGS sequencing can help us comprehend DNA modification that affect the expression pattern of the given genes (epigenetic profiling) as well as characterizing the DNA-protein interactions that allow for the identification of genes that may be regulated by a given protein.  DNA Methylation Profiling…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.