by Caitlin Smith
Beam me up, Scotty? We’re not quite there yet, but today scientists are using lasers to cut, capture, and even catapult single cells and pieces of tissue. Laser capture microdissection (LCM) uses inverted or upright microscopes, ultraviolet (UV) or infrared (IR) lasers, and tissue from a variety of sources.
Until recently, laser capture microdissection was mostly limited to samples such as cryosections, or formalin-fixed paraffin-embedded (FFPE) materials. For example, you can isolate rare cell populations in culture, or remove malignant cells from cancerous tissue for closer study in primary culture, investigating with pharmacological evaluation or analysis of single-cell genetic complements or proteomes. Isolating differentiated or undifferentiated stem cells may also become a more common application of laser capture microdissection in the future – one of many. “The flexibility and utility of laser capture microdissection as a sample preparation tool has lent itself to a diverse set of research areas and applications, including cancer biology, neurobiology, and developmental biology,” says Steven Blakely, product manager for Arcturus LCM instruments and reagents at MDS Analytical Technologies, “and it is exciting to see the technology expanding into new areas, such as live cell research, forensics sciences, plant biology and drug discovery.”
Innovations in cutting
Despite the precision achieved with lasers to cut tissue, the laser beam itself has a width that can damage surrounding tissue, and potentially even your sample of interest. Molecular Machines and Industries (MMI) has developed an ultra fine cutting laser. MMI’s high speed UVa laser is focused through the 100x objective, giving a 0.3 um width. The dissected tissue is then lifted away on an adhesive cap. “Our latest developments lie in the area of single cell analysis,” says Antje Plaschke-Schlütter, head of business development and applications at MMI. “We combine on the same microscopic platform laser-based and capillary-based, fully automated single cell selection technologies. Our CombiInstrument CellCutplus and CellEctor meet the particular needs of researchers and medical doctors who want to isolate cells from tissue and suspension, e.g. a biopsy and blood or bone marrow of the same patient.” Plaschke-Schlütter sees a challenge ahead in establishing “laser microdissection and subsequent molecular profiling in personalized medicine.”
Leica Microsystems has developed two systems for laser capture microdissection. “We offer two models with different UV lasers for microdissection – a cold ablation cutting method,” explains Ted Morris, product marketing manager of research microscopy in the life science division at Leica Microsystems. “The LMD 6500 has a 355 nm laser with a power of >50 uJ for most biological samples. The LMD 7000 uses a more powerful 120 uJ laser at 349 nm and has an innovative ability to control not only the power but also the repetition rate. This model is ideally suited for rapid cutting of hard tissue samples, bone, tooth enamel, and plant.” They use an upright research microscope with an optically moveable laser to follow the cut pattern drawn by the laser. “The dissectate drops into a tube cap placed just below the specimen for a contamination-free collection,” says Morris. “Others use an inverted microscope with a fixed laser position and move the sample around during cutting via a motorized stage to follow the drawn pattern. These inverted microscopes rely on pushing the dissected area into a sticky cap or melted foil to pull off the selected cells.”
Morris sees an upcoming challenge in transforming single cell analysis into a high-throughput process: “After microdissection, the downstream analysis step is next, and this is where the amplification and analysis of macro molecules (DNA, RNA, proteins) in one single cell is challenging. Scientists must therefore collect a high volume of dissected samples for analysis - a time consuming task. Developments in the future would be to link laser microdissection with fast slide scanners or tissue microarray analyzers. This speeds up the process since scanners can identify about 60% of the markers in tissue to do further analysis on and laser microdissection. If the throughput can be increased then the laser microdissection could move into the clinical diagnostic field.”
To eliminate the complications of cross-contamination that can occur when your excised sample is touched upon removal, Zeiss developed a dissection system featuring contact-free sample manipulation. Their PALM MicroBeam with inverted microscope outlines the area of excision with a UV laser. Then it uses a defocused laser pulse to create a gaseous expansion beneath the excised tissue, which accelerates (or catapults) the sample into the collection vessel – an inverted microfuge tube cap above the sample. “First and foremost the purity of the isolated sample is really possible due to our catapulting technology against gravity using a forceful pulse to propel the sample to an awaiting capture device above the sample that never touches and thus disturbs the sample,” says Timothy Pratt, imaging application support and LCM support specialist at Zeiss. “Additionally, our system stands out from our competitors because of its power as a superior imaging device possible across many different imaging and contrast methods.”
New releases from Zeiss include instrumentation to enhance imaging functionality on their LCM systems using their AxioObserver inverted microscope, which also functions as a non-LCM scope. “This inverted scope platform offers both fixed and live cell possibilities including live cell incubation chambers for long-term time-lapse work and both positive and negative sample isolation,” says Pratt. “Now we offer true confocal technology with our [LCM] systems. Thus, users can have rapid confocal imaging technology possible for their sample identification, with superior axial resolution for accurate sample selection combined with state-of-the-art laser manipulation.” For live cell work, they also offer a single-cell collection system using a new capture plate, the SlideCollector 48, for downstream PCR analysis.
Greater automation will become more important in LCM workflows in the future, according to Pratt: “More and more there is a need for high degrees of automation, creating hurdles in affordable and flexible instrumentation, while reducing risks for loss of sample integrity by making faster and more precise isolation systems while using the procedure of LCM. Scientists have a certain sample capacity; LCM must allow for that and should not be the bottleneck. More automation would allow for everything from rapid positive cell identifications to capture for all sorts for downstream nucleic acid and protein analyses.”
Capturing live cells
Laser capture microdissection is also an important tool for the isolation of live cells – for example, in choosing cells from cancerous tissue to grow in primary culture. MDS Analytical Technologies recently released the Live Cell Application Module for their ArcturusXT™ Laser Capture Microdissection instrument. “Using a custom-designed modular stage insert, the ArcturusXT instrument may be used for imaging and microdissection of living cells from a Petri dish format,” says Blakely. “We have developed and validated a protocol to isolate live cells while maintaining cell viability for subsequent re-culture or for direct processing for use in downstream genomic or proteomic applications.” The ArcturusXT uses infrared laser-based capture technology to collect samples. “Whether researchers are using IR capture alone or in combination with the optional ultraviolet laser cutting, the gentle, non-damaging, IR capture technology allows the custody of the microdissected sample to be maintained throughout the process,” says Blakely.
Blakely notes that another exciting area of growth for laser capture microdissection is in forensic sciences. “For example, laser capture microdissection can be used to help identify a perpetrator in a rape case,” says Blakely. “When a sample is taken from a rape victim, there may be only a few sperm among thousands of epithelial cells from the victim. Separation of the sperm from the epithelial cells is needed for accurate DNA analysis of the sperm, but traditional separation methods may struggle to effectively isolate those limited number of sperm.”
Processing microdissection samples before and after the laser capture event itself continues to challenge scientists, says Blakely. “For instance, if handled improperly, biomolecules can degrade due to enzyme activity, which would make downstream processing difficult or impossible,” he explains. “Downstream processing is also a challenge for some researchers due to the very limited amount of material that is collected using LCM. Some researchers are collecting only a few cells, maybe only one cell, and so they need to make sure that those cells are not damaged in the LCM process. MDS Analytical Technologies recognizes this challenge and offers a full system of instruments, reagents and protocols, all devised specifically for the processing of samples for LCM and for the preservation of biomolecules for downstream processing.”
One day, microdissection may become a completely automated procedure to enable high throughput sorting of stem cells, or primary cell types from tissue samples, for example. In the meantime, individual scientists still need to apply their own criteria in deciding which cells to beam up.