Laser Capture Microdissection

Specifications

The PixCell II ™ Laser Capture Microdissection system with epifluorescence was developed by Arcturus Engineering in collaboration with the National Cancer Institute of the NIH. The PXL-200 laser capture system includes: a solid state near infrared laser diode with <7.5 µm, 15 µm, and 30 µm 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. The ARC-200 image archiving workstation consists of: the ARC-200 IA software from Arcturus;a Dell 4.3 GB Pentium III with a CD-ROM drive; and a Sony Trinitron monitor. A grant application has been submitted to upgrade to the new ArcturusXT instrument described on the Molecular Devices website.

Examples of single rat brain cells (upper panels) that have been laser captured at this facility. Success is measured by what you see on the cap!

Procedures

Pure samples of cells captured by LCM are today’s standard for accurate, reliable and reproducible results in Genomics, Transcriptomics, Proteomics, and Metabolomics. You can precisely identify and specifically select individual cells or groups of cells from tissue sections, blood smears, or cell cultures. LCM optimizes precision measurements of DNA, mRNA, or protein by providing pure cell samples essential for all molecular analysis.
Cell procurement must be made from a dehydrated tissue section or cell sample centered on a glass slide. The CapSure Cap, an optically transparent cap containing a thermoplastic membrane transfer film, is placed on top of the slide and cells are dissected by the focal melting of the membrane through laser activation. This short, low power infrared laser pulse, with beam sizes of 7.5 mm, 15 µm and 30 mm, is triggered with a push of a button. 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 of the captured cells on the CapSure cap.
Detailed procedures for specific applications currently can be found on the Molecular Devices/Arcturus site. In addition, the LCM facility will work closely with each investigator to develop optimal procedure(s) for tissue preparation specific to each sample.

Examples of single rat brain cells (upper panels) that have been laser captured at this facility. Success is measured by what you see on the cap!

Policies

  1. Each scientist, physician, or pathologist who wishes to use the LCM system must first be trained and certified by Dr. Hao Chen hchen@uthsc.edu.
  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 at www.moleculardevices.com/pages/instruments/arcturusXT.html.
  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:
    1. any special cell or tissue stain(s) other than Nissl or H&E; Molecular Devices has kits available for:
      • Staining frozen samples
      • Immunofluorescence
      • RNA isolation
      • DNA isolation
      • RNA amplification
      • RNA amplification-high sensitivity
      • Formalin-fixed paraffin-embedded sections
      • Microarray labeling kits
    2. providing your own CapSure caps
    3. specific extraction buffers in microfuge vials for each sample;
    4. performing all subsequent assays.
    5. removing all digitized images from the computer at the end of each session. These images can be downloaded onto a CD or RAM disk. The LCM computer files will be purged on a regular basis in order to insure smooth operation of the image intensive program(s). Therefore, if you fail to remove your digitized images to another source for archival purposes, you are not allowed to complain that the system is running too slowly!
  5. It has rapidly become apparent that prolonged, continuous use of the LCM is counterproductive. The procedure requires concentrated focus and attention - pushing yourself (or your graduate students and post-docs) for hours at a time invariably results in mistakes and/or poorly captured cells. Therefore, 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.

Laser Capture Microdissection Facility

Hao Chen, Ph,D.
Director
Assistant Professor
Department of Pharmacology
Nash Annex, Room 250F
Memphis, TN 38163
Phone: 901-448-3720
Email: hchen@uthsc.edu