The Robot-Microbeam technology for single cell preparation
and laser microinjection
Schütze K., Lahr, G., Stich, M. and Pösl, H..
Applikatives Laserzentrum der I. Medizinische Abteilung, Krankenhaus Harlaching,
D-81545 München, Germany
The procurement of single cells from different sources for subsequent genetic analysis increasingly gains importance in a wide field of cell biology and molecular medicine.
The Robot-Microbeam (P.A.L.M. GmbH, Wolfratshausen) consists of a 337nm nitrogen laser, coupled into a microscope and focused through high numerical objectives to a diameter of less than one micrometer. The high photon density within the focal point is used to cut out tissue areas or single cells (Laser microbeam microdissection – LMM). The focus of the laser beam can precisely follow the irregular shape of the specimen. With a few laser shots the laser-isolated specimens are catapulted away from the object slide directly into the cap of a microfuge tube or onto a small piece of glass (Laser pressure catapulting – LPC) [1].
The Robot-Microbeam was used to procure a few cells from a colon adenocarcinoma. RT-PCR amplification of m RNA for cytoplasmic ß-actin was not impaired by laser treatment. The primer sets were designed to span introns to eliminate any potential signal from contaminating chromosomal DNA. Thus, neither the routine histological treatment of the tumor slices, like paraffin embedding, nor LMM or LPC impairs mRNA analysis.
Bone marrow of a patient with adenocarcinoma was screened for epithelial cells, known as micrometastases. Cells were cytocentrifugated onto glass slides and immunohistochemically stained for cytokeratines according to standard methods. Micrometastases were selectively isolated with the Robot-Microbeam for further biomolecular analysis.
Contact-free laser microinjection can be performed with the same system poking minute holes into living cells. Genetic material or substances present within the culture medium are simply swallowed by the microperforated cells before the holes are sealed again by cell reparation mechanisms.
In summary, the Robot-Microbeam technology is a valuable tool for a variety of applications like genetic engeneering or where the procurement of single cells or specific cell clusters are required for further genetic and molecular biological analysis [2].
[1] Schütze, K., Becker, I., Becker, K.-F., Thalhammer, S., Stark, R., Heckl, W.M., Böhm, M., & Pösl, H. Cut out or poke in - the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease. Genet. Analysis: Biomol. Engineering 14, 1-8 (1997)
[2] Böhm, M., Wieland, I., Schütze, K., & Rübben, H. Micobeam MOMeNT non-contact laser microdissection of membrane mounted native tissue. Am. J. Pathol. 151, 63-67 (1997)
| LOCATION |
DATE |
TIME |
| Lecture Hall II |
Sunday, April 5 |
03:20 pm |