Welcome to Laser Capture Microdissection

Laser Capture Microdissection (LCM) provides a rapid, reliable, one-step method to obtain pure populations of targeted cells from specific microscopic regions for subsequent analysis. LCM was developed to enable investigators and clinicians to perform tissue microdissection on a routine basis. There are no limitations in the ability to amplify DNA or RNA from individual cells or cell clusters (e.g., tumors) from tissue sections, blood smears, or cell cultures. LCM has been used to explore gene expression, loss of heterozygosity, micro-satellite instability, clonality and for proteomics. The Arcturus Pixcell II LCM at the UTHSC facility has major advantages compared to other instruments in that 1) the laser never touches the tissue itself, thereby avoiding possible heat shock and damage, 2) cells are captured directly onto an optically transparent cap for reliable transfer of every sample to the a ppropriate assay tube, and 3) the accuracy of each step of cell capture can be digitally documented by the accompanying archiving software. The PixCell II also has a fluorescence system with filter cubes for blue (Ex 455-495 nm/Em >510 nm), green (Ex 503-547 nm/Em >565 nm), and red (Ex 590-650 nm/Em >667 nm) wavelengths. The following fluorophores have been shown to be compatible with LCM: Cy2, Cy3, Cy5; Alexa 488, 532, 546, 568; FITC; Oregon Green; propidium iodide; TRITC; phycoerythrin; rhodamine; To-Pro-3; and, Texas Red.

Procedures

Cells can only be captured from dehydrated tissue sections or cell samples centered on a glass slide. The proprietary CapSure, an optically transparent cap containing a thermoplastic membrane transfer film, is placed on the slide and cells are dissected by the focal melting of the membrane through laser activation (fig 1). This short, low power infrared laser pulse, with beam sizes of 7.5, 15 and 30 μm, is triggered with a push of a button on the joystick. At no time does the laser directly touch the tissue sample; therefore, neither the quality of nucleic acids and proteins within the sample nor cell morphology are compromised, and the surrounding tissue remains intact on the slide. The captured cells remain attached to the transfer film surface on the CapSure cap, which is then placed directly into a microcentrifuge tube containing the user identified extraction buffer for specific assay(s). Publication quality, digitized images can be taken of the tissue section before and after LCM, as well as of the selected tissue retained on the CapSure cap, to document the specifi city of each sample (fig. 2). Extraction of DNA has been demonstrated using both ethanol fixed and formalin fixed paraffin embedded tissue; RNA can be recovered from fresh frozen tissue sections. Guidelines for amounts needed per sample for molecular analyses are: 10-20 cells from a 10-micron section has been shown to work for PCR; 50,000 cells for 2-D gel a nalysis; and, 20,000 cells for a western blot.

Since slide preparation is the most critical component of successful laser capture, the Director of the LCM facility, Dr. Shannon Matta, will work closely with each investigator to develop optimal procedure(s) for tissue preparation specific to the sample. In addition, every scientist, physician or pathologist who wishes to use the LCM system must first be trained and certified by Dr. Matta.

For more information, see the laser capture microdissection facility homepage at www.utmem.edu/lcm.

Contact Us

Shannon Matta, Ph.D., Director
Pharmacology
(901) 448-2874
(901) 448-6000 (alternate)
Email: smatta@utmem.edu