The Laser Capture Microdissection (LCM) is a university-wide core facility on the campus of the University of Tennessee Health Science Center in Memphis, TN. 

         This facility is directed by Dr. Shannon Matta smatta@utmem.edu .

  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; blue  filter 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 3.5" floppy, ZIP, and CD-ROM drives; and a Sony Trinitron monitor. The system is connected to the University network via a T10 line and images can be downloaded directly into the electronic drop-box of each investigator's computer.

  Procedures

Pure samples of cells captured by LCM are today’s standard for accurate, reliable and reproducible results in Genomics and Proteomics. 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 (see: LCM video). 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 Arcturus site www.arctur.com. In addition, the LCM facility will work closely with each investigator to develop optimal procedure(s) for tissue preparation specific to each sample.

  Policies

1.    Each scientist, physician, or pathologist who wishes to use the LCM system must first be trained and certified by Dr. Shannon Matta smatta@utmem.edu .

2.    Currently, there is an $18 hourly charge for using the LCM. There is no charge for training.  Disposable LCM supplies (see below) will be provided for training, after which you will be responsible for purchasing your own.

  3.    Since slide preparation is the most critical component of successful cell capture, it is strongly recommended that each investigator initially work with Ms. Spencer 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 adjacent LCM wet lab (250E Nash Annex) 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:

        A) any special cell or tissue stain(s) other than Nissl or H&E;

        B) specific extraction or dilution buffer(s);

        C) performing all subsequent assays.

        D) removing all digitized images from the computer at the end of each session. These images can be downloaded onto a ZIP disk or sent directly to the investigator's drop-box via the network. 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 student) 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. Matta. 

  LCM Supplies  (2001-2002 prices from Arcturus) 

*These are the standard LCM caps for general work and for capture of a large number of cells.

** These are the high sensitivity caps that work best for capturing a small number of single cells. They are used in conjunction with the ExtracSure  device, especially when smaller volumes of buffer are necessary. The ExtracSure device comes with the Capsure HS caps or can be purchased separately.

Supplies may be purchased from Arcturus by phone order (only with a credit card), FAX, or e-mail (with a P.O.#):  

            Arcturus

            400 Logue Ave.

            Mountain View, CA 94043

            Tel: 650.962.3020 or toll free 888.446.7911

            Fax: 650.962.3039

            Federal ID #: 77 0434959

            Email: contact@arctur.com

            Internet access: www.arctur.com

Other recommended items:

1. Microcentrifuge tubes: Applied Biosystems GeneAmp™ 500 ul Thin-walled  PCR Reaction Tubes, autoclaved: cat# 9N801-0611. Non-autoclaved: cat# N801-0537. Currently, these are the only known tubes that fit the LCM caps. To order call 1.800.345.5224 or fax 650.638.5884.

2.  ZIP disk(s) (if you choose not to use direct electronic transfer via the network). Be aware that, depending on the size of each image, a ZIP disk can usually hold only 6-10 images.

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