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Laser Capture Microdissection Facility

The Laser Capture Microdissection (LCM) Facility serves the campus of the University of Tennessee Health Science Center in Memphis, Tennessee.

LCM optimizes the precision measurements of DNA, mRNA, or protein by providing cell samples collected under a microscope. These cells can be identified and collected from tissue sections (stained or unstained), blood smears, or cell cultures.

How does LCM work?

Laser Capture Microdissection is a new technology developed to enable investigators and clinicians to perform tissue microdissection on a routine basis. The instrument was conceived as a prototype research tool at the NIH. It was then developed through a cooperative research and development agreement (CRADA) between Arcturus Engineering and NIH, NCRR, and NCI.

LCM provides a rapid, reliable, one step method to procure pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis. A thermoplastic polymer used on a cap (see diagram below) bonds to the tissue after being targeted with the laser. This avoids chemical reactions that can cross-link biological molecules in the tissue and alter subsequent molecular analysis. LCM has successfully extracted cells in all tissues in which it has been tested. In addition, there are no limitations in the ability to amplify DNA or RNA from tumor cells. LCM can capture cells without damaging the enzymatic function of the proteins.

Extraction of DNA has been demonstrated using both ethanol fixed and formalin fixed paraffin embedded tissue. RNA can be recovered by dissecting fresh frozen tissue sections. Some guidelines for tissue needed for molecular analysis: 10-20 cells from a 10-micron thick PET section has been shown to work for PCR, 50,000 cells for a 2-D gel analysis and 20,000 cells for Western Blot. LCM has been used to explore gene expression, loss of heterozygosity, micro-satellite instability, clonality and in proteomics.

diagram

The LCM instrument is available for your use in the Translational Science Research Building, Room 230L. Please contact Dr. Hao Chen - 448-3720 for information on training sessions.

Policies
  1. Each scientist, physician, or pathologist who wishes to use the LCM system must first be trained and certified by Dr. Hao Chen.
  2. Currently, there is a $26 hourly charge for using the LCM, including training time. Disposable LCM supplies will be provided for training, after which you will be responsible for purchasing your own from LifeTechexternal link icon
  3. Since slide preparation is the most critical component of successful cell capture, it is strongly recommended that each investigator initially work with Dr. Chen to optimize tissue preparation procedures specific for the user-defined tissue samples prior to attempting LCM on critical experimental or clinical samples. Once such a protocol has been optimized for each user, individual researchers can opt to continue slide preparation in the LCM wet lab or can prepare slides in his/her lab. In the latter case, a small dessicator or slide box containing dessicant will be necessary for transporting the slides to the LCM facility from other laboratories.
  4. Individual researchers are responsible for:
    • any special cell or tissue stain(s).
    • specific extraction buffers in microfuge vials for each sample;
    • performing all subsequent assays.
    • removing all digitized images from the computer at the end of each session.
  5. The use of the LCM facility is limited to a maximum of 3 hours in any one session. Sessions can be scheduled from 9 AM - 12 noon (first session) or 1 PM - 4 PM (second session). No evening hours, weekends or holidays will be available without extenuating circumstances and require prior arrangement with Dr. Chen.
Instrument
The PixCell II ™ Laser Capture Microdissection system includes: a solid state near infrared laser diode with <7.5 µ m, 15 µ m, and 30 µ m spot sizes; the Controller power supply (0-100 mW maximum); an Olympus IX70 inverted microscope with 4x, 10x, 20x, and 40x objectives; an Hitachi KPD-580 color CCD video camera; a Matrox Meteor™ color frame grabber board; and a Sony Trinitron video monitor. The Fluor-300 fluorescence system is comprised of: a 100W mercury arc lamp; bluefilter cube (Ex 455-495 nm/Em >510 nm); green filter cube (Ex 503-547 nm/Em >565 nm); red filter cube (Ex 590-650 nm/Em >667 nm); and an IR blocking filter for the camera and a Sony Trinitron monitor.

The Arcturus ® XT system include: a Nikon Eclipse® Ti-E microscope base; infrared (IR) Capture Laser: Solid-state, near-IR (810 nm); UV Cutting Laser: Solid-state, passive Q-switched, diode-pumped (355 nm) ; 2x, 10x, and 40x Nikon CFI60 objectives ; motorized stage with 1 µ m precision, and the up-to-date software.

More system details available at the LifeTech websiteexternal link icon

May 26, 2022